Multiple climbing fiber innervation of Purkinje cells in the adult cerebellum

大脑回路的结构在理解其功能方面起着至关重要的作用。的发现 生理学和神经计算中的主要概念严重依赖于精确的 形态学知识支持这一观点的一个突出例子是小脑，一种大脑回路， 参与运动（和非运动）控制和适应。小脑回路的中心是浦肯野细胞， 投射神经元接收数千个兴奋性突触，这些突触传递所需的感觉信息。 来控制运动神经这些独特的神经元的一个标志性特征是它们有一个巨大的树突， 在成年人的大脑中，是由一个攀爬纤维输入来支配的。攀缘纤维在植物生长理论中起着至关重要的作用 小脑监督学习，并在这个概念框架内提供了一个指导性的信号（在这里， 误差信号）在平行纤维突触处引导可塑性。此外，最近的研究还指出， 在奖赏信号中的作用，并指出攀爬纤维诱发的复杂尖峰是对各种 感觉刺激，并且还可以携带运动命令信号。这些活动依赖的复合峰是 观察到除了自发复杂的尖峰，发生在平均频率约为1Hz。因此，在本发明中， 虽然我们不知道攀爬纤维信号的确切功能是什么，但很明显， 输入提供了小脑皮层回路的核心元素，并在某种程度上定义了其操作。我们 发现-这里作为试点数据-整个成人浦肯野细胞群的一个亚群（约15%） 有两个攀爬纤维输入，而不是只有一个，这两个输入几乎只在浦肯野观察到 具有两个初级树突的细胞（分别从索马伸出，或在其附近分离） 在这个亚组中，20-25%的浦肯野细胞显示两种攀爬纤维输入，这导致了以下问题 这种双重神经支配是否具有功能意义。是持久的双攀缘纤维 成年浦肯野细胞的神经支配是一个错误还是一个特征？在这项探索性研究中，我们计划研究 进一步分析基本现象，并评估两个单独的攀爬纤维输入是否可以独立地发信号。 如果我们的“缺陷或特征”问题的答案是“特征”，我们将检查小脑功能的后果 (e.g.行为学习测试）在随后的研究中，我们将寻求单独的资金。第一个目标是使用 在体外结合共聚焦显微镜的小鼠膜片钳记录来检验假设， 多个攀缘纤维的持续存在不是随机的，而是发生在具有两个初级纤维的浦肯野细胞中。 树突我们还将在下橄榄注射示踪染料，染色攀爬纤维， 存在两个爬升纤维输入。第二个目标将增加体外钙成像，以检查是否 双攀爬纤维输入导致功能上分离的树突状钙信号传导结构域。第三个目标 使用清醒小鼠浦肯野细胞的基于GCaMP 6 f的双光子成像， 在自发条件下，或在感觉刺激下，两个攀爬纤维输入可以独立地操作。

The architecture of brain circuits plays an essential role in understanding their function. The discovery of principal concepts in physiology and neurocomputation critically depends on the availability of precise morphological knowledge. A prominent example supporting this notion is the cerebellum, a brain circuit that is involved in motor (and non-motor) control and adaptation. At the center of cerebellar circuits are Purkinje cells, projection neurons that receive thousands of excitatory synapses, which convey sensory information needed for proper motor control. A hallmark feature of these unique neurons is that they have a massive dendrite that is innervated, in the adult brain, by one climbing fiber input. The climbing fiber plays a crucial role in theories of cerebellar supervised learning, and within this conceptual framework provides an instructive signal (here an error signal) guiding plasticity at parallel fiber synapses. More recent studies have, in addition, pointed out roles in reward signaling and noted that climbing fiber-evoked complex spikes occur in response to a variety of sensory stimuli and may also carry motor command signals. These activity-dependent complex spikes are observed in addition to spontaneous complex spikes that occur at an average frequency of about 1Hz. Thus, while we do not know what the exact functions of climbing fiber signaling are, it is obvious that this unusual input provides a core element of cerebellar cortical circuits and to some degree will define its operations. Our discovery – presented here as pilot data – that a subgroup of the entire adult Purkinje cell population (~15%) has two climbing fiber inputs instead of just one, that two inputs are almost exclusively observed in Purkinje cells with two primary dendrites (either separately exiting from the soma, or separating in close proximity to it) and that within this subgroup 20-25% of Purkinje cells show two climbing fiber inputs, leads to the question whether there is a functional significance of this double innervation. Is persistent double climbing fiber innervation of adult Purkinje cells a bug or a feature? In this exploratory study, we plan to examine the basic phenomenon further and to assess whether two separate climbing fiber inputs may signal independently. If the answer to our ‘bug or feature’ question is ‘feature’, we will examine consequences for cerebellar function (e.g. behavioral learning tests) in subsequent studies that we will seek separate funding for. The first aim uses patch-clamp recordings from mice in combination with confocal microscopy in vitro to test the hypothesis that the persistence of multiple climbing fibers is not random, but occurs in Purkinje cells with two primary dendrites. We will also inject tracer dyes into the inferior olive to stain climbing fibers and anatomically confirm the existence of two climbing fiber inputs. The second aim will add calcium imaging in vitro to examine whether a double climbing fiber input leads to functionally separate, dendritic calcium signaling domains. The third aim uses GCaMP6f-based two-photon imaging from Purkinje cells in awake mice to assess whether under spontaneous conditions, or with sensory stimulation, two climbing fiber inputs may operate independently.