Neuronal plasticity and cerebellar circuitry

描述（由申请人提供）：小脑回路的体内分析几乎完全集中在浦肯野细胞上，通过其复杂和简单尖峰（CSs和ss）的标志性模式来识别。然而，完全基于浦肯野细胞生理学的小脑功能观点忽视了中间神经元的作用，扭曲了小脑传入系统的属性。普遍认为，SSs是由苔藓纤维-颗粒细胞平行纤维投射到浦肯野细胞树突的活性调节的。事实上，在自然的前庭刺激过程中，前庭初级传入苔藓纤维传入与结节性浦肯野细胞记录的SSs不同步放电。因此，小脑输出信号不太可能仅仅反映苔藓纤维输入信号的增益控制版本。我们认为SS调制反映了攀爬纤维对小脑中间神经元的作用。我们将通过记录已识别的中间神经元来检验这一假设的特定版本。我们有三个目标。首先，我们将记录麻醉小鼠在自然前庭刺激时小舌结节中间神经元的细胞外活动。中间神经元将在细胞旁用神经生物素标记。相对于浦肯野细胞CSs和SSs，神经元间放电调节的深度和相位将表明哪些中间神经元可以调节SSs。其次，我们将研究单侧迷路切除术（UL）如何改变中间神经元和浦肯野细胞的调节活性。在UL后，同侧小舌结节只能通过攀爬纤维接收前庭信息，攀爬纤维的调节依赖于对侧完整的迷路。第三，我们还将在对侧下橄榄的p核和背内侧细胞柱（dmcc）中进行微病变。这将使小脑的一侧仅通过前庭苔藓纤维接收前庭信息。我们将比较前庭信号减少对中间神经元和浦肯野细胞的影响。第四，我们将在带有细胞特异性启动子的病毒载体中微注射mirna，选择性地降低结核性浦肯野细胞中GABA-A α 1受体的表达。第五，我们还将利用miRNAs选择性地减少高尔基细胞中GABA的合成。我们将分析“敲低”gaba能信号在这两种细胞类型中的作用。我们将首次描述已识别的中间神经元的刺激调节功能。我们将在细胞水平上干扰小脑回路，并测试中间神经元在SSs调节中的作用。提出的研究将加速分子技术在小脑疾病患者治疗中的应用。

DESCRIPTION (provided by applicant): In vivo analysis of cerebellar circuitry has focused almost exclusively on Purkinje cells, identified by their iconic patterns of complex and simple spikes (CSs and SSs). However, views of cerebellar function based exclusively on the physiology of Purkinje cells ignore the role of interneurons and distort the attributes of cerebellar afferent systems. It is universally assumed that SSs are modulated by the activity of the mossy fiber-granule cell-parallel fiber projection to Purkinje cell dendrites. In fact, during natural vestibular stimulation, vestibular primary afferent mossy fiber afferents discharge out of phase with the SSs recorded from nodular Purkinje cells. Consequently, it is unlikely that the cerebellar output signal merely reflects a gain-controlled version of the mossy fiber input signal. We proposed that SS modulation reflects the action of climbing fibers on cerebellar interneurons. We will test specific versions of this hypothesis by recording from identified interneurons. We have three objectives. First, we will record extracellularly from interneurons in the uvula-nodulus of anesthetized mice during natural vestibular stimulation. Interneurons will be labeled juxtacellularly with neurobiotin. The depth and phase of modulation of interneuronal discharge relative to that of Purkinje cell CSs and SSs will indicate which interneurons could modulate SSs. Second, we will study how the modulated activity of interneurons and Purkinje cells is altered by a unilateral labyrinthectomy (UL). Following a UL, the ipsilateral uvula-nodulus is accessible to vestibular information mediated only by climbing fibers whose modulation depends on the contralateral, intact labyrinth. Third, we will also make microlesions in the p-nucleus and dorsomedial cell column (dmcc) in the contralateral inferior olive. This will leave one side of the cerebellum accessible to vestibular information mediated only by vestibular mossy fibers. We will compare the effects of reduced vestibular signaling on interneurons and Purkinje cells. Fourth, we will microinject miRNAs in viral vectors with a cell specific promoter to selectively reduce expression of GABA-A alpha 1 receptors in nodular Purkinje cells. Fifth, we will also use miRNAs to selectively reduce synthesis of GABA in Golgi cells. We will analyze the effects of "knocking down" GABAergic signaling in these two cell types. We will characterize the stimulus-modulated functions of identified interneurons for the first time. We will interfere with cerebellar circuitry at a cellular level and test the role of interneurons in the modulation of SSs. The proposed research will speed application of molecular techniques to the treatment of patients with cerebellar disorders.