Multiple climbing fiber innervation of Purkinje cells in the adult cerebellum

大脑回路的结构在理解它们的功能方面起着至关重要的作用。发现了 生理学和神经计算的主要概念在很大程度上依赖于 形态知识。支持这一概念的一个突出例子是小脑，这是一个大脑回路 参与运动(和非运动)控制和适应。小脑环路的中心是浦肯野细胞， 接受数千个兴奋性突触的投射神经元，这些突触传递所需的感觉信息 以进行适当的运动控制。这些独特神经元的一个显著特征是它们有一个巨大的树突 在成人大脑中，由一个攀爬纤维输入来神经支配。攀爬纤维在生物力学理论中起着至关重要的作用。 小脑监督学习，在这个概念框架内提供了一个有指导意义的信号(这里是 错误信号)引导平行纤维突触的可塑性。此外，最近的研究还指出， 在奖赏信号中的作用，并注意到攀登纤维诱发的复合峰发生在对各种 感官刺激，也可以携带运动指令信号。这些依赖于活动的复杂尖峰是 除了以大约1赫兹的平均频率出现的自发的复杂尖峰电位外，还可以观察到。因此， 虽然我们不知道攀升光纤信号的确切功能是什么，但很明显，这一不寻常的 输入提供了小脑皮质回路的核心元素，并在某种程度上定义了其运作。我们的 发现--这里作为试点数据--整个成年浦肯野细胞群体的一个亚群(~15%) 有两个攀升光纤输入，而不是只有一个，这两个输入几乎只在浦肯野观察到 有两个初级树突的细胞(要么从胞体分开，要么在胞体附近分离) 在这个亚群中，20%-25%的浦肯野细胞显示出两个攀升的纤维输入，这引发了这个问题 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种缺陷还是一种特征？在这项探索性研究中，我们计划研究 进一步了解基本现象，并评估两个单独的上升光纤输入是否可以独立地发出信号。 如果我们的“缺陷或功能”问题的答案是“功能”，我们将检查对小脑功能的影响 (例如，行为学习测试)在后续研究中，我们将寻求单独的资金。第一个目标是使用 小鼠的膜片钳记录与体外共聚焦显微镜相结合来验证这一假说 多个爬行纤维的持续不是随机的，而是发生在具有两个初级细胞的浦肯野细胞中 树枝状结构。我们还将在下橄榄内注射示踪染料以染色攀升纤维并在解剖上确认 存在两个爬升光纤输入。第二个目标是在体外添加钙成像，以检查 双爬入纤维导致功能分离的树突状钙信号结构域。第三个目标 使用基于GCaMP6f的双光子成像技术从清醒小鼠的浦肯野细胞中评估在 在自发情况下，或在有感觉刺激的情况下，两个攀升纤维输入可能独立运行。

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.