Optogenetic investigation of olivary inputs to the deep cerebellar nuclei

描述(申请人提供)：小脑是一个大的后脑结构，涉及运动控制的许多方面以及行为的认知方面。小脑功能障碍已被确认为重要运动障碍的主要原因，特别是共济失调和某些形式的肌张力障碍。然而，从根本上讲，我们并不了解小脑是如何运作的。这在一定程度上是因为缺乏适当的实验技术来操纵小脑网络的不同亚单位来研究其功能。遗传小鼠品系开始帮助分离与疾病有关的特定小脑功能，目前的研究正在使用小鼠与这项研究相联系。本方案的具体创新包括使用一种新开发的光遗传学方法，该方法允许将通道视紫红质-2(ChR2)基因插入大脑的特定区域或细胞类型。ChR2是一种光敏离子通道，当暴露在蓝色波长的光下时，它会导致去极化电流进入表达它的神经元。我们将通过腺相关病毒载体注射在成年小鼠的下橄榄中表达ChR2，然后研究光刺激小脑中橄榄轴突向小脑的橄榄输入。这是我们解剖小脑回路能力的一个重要进步，因为以前电刺激只允许各种纤维通路的混合激活。最重要的是，提供小脑最终输出的小脑深核(DCN)接受来自橄榄轴突的兴奋性输入，但这一通路的这一功能意义从未被分离出来。相比之下，小脑皮质中的浦肯野细胞以攀爬纤维的形式接受橄榄糖输入，这已经得到了广泛的研究，因为每一个单独的输入都非常强大，很容易被识别为攀爬纤维反应。我们将使用大脑切片记录和来自小脑网络完整的动物的记录来确定向DCN输入橄榄的效果。切片记录将使我们第一次确定橄榄核与DCN神经元联系的详细突触属性。来自完整动物的录音将阐明这种连接在完整网络中的影响，以及是否相同的橄榄轴突刺激DCN神经元也会通过激活浦肯野细胞攀升纤维而导致同一神经元的延迟抑制。总体而言，我们的光遗传学研究解决了一条重要的小脑输入通路的作用，到目前为止，这一通路还不能用经典技术进行研究。我们预计将发现对浦肯野细胞输入的DCN平衡抑制作用有显著的兴奋作用。这一认识将为未来研究橄榄糖输入通路在小鼠共济失调和肌张力障碍突变体的小脑疾病状态中的参与奠定基础。 与公共卫生相关：小脑是致衰性运动障碍的来源，最明显的是小脑性共济失调和某些形式的肌张力障碍。为了更好地理解和治疗这些疾病，我们将用光遗传学方法研究对小脑深部核团的兴奋性输入，从而首次详细分析这一途径。这一分析将有助于我们在未来的研究中检查导致小脑疾病的小鼠突变体小脑活动的变化。PHS 398/2590(06/09版)页面续格式页面

DESCRIPTION (provided by applicant): The cerebellum is a large hindbrain structure involved in many aspects of motor control as well as cognitive aspects of behavior. Dysfunction of the cerebellum has been identified to be the primary cause in important motor disorders, notably ataxias, and some forms of dystonia. Nevertheless, at a fundamental level we do not understand how the cerebellum operates. This is in part due to a lack of the appropriate experimental techniques that can manipulate different subunits of the cerebellar network to study its function. Genetic mouse lines are starting to help in isolating specific cerebellar functional involvement in disease, and the present study is using mice to link to this research. The specific innovation of the present proposal consists of using a newly developed optogenetic approach that allows the insertion of channelrhodopsin-2 (ChR2) gene into specific areas or cell types of the brain. ChR2 is a photosensitive ion channel that results in a depolarizing current into neurons expressing it when exposed to blue-wavelength light. We will express ChR2 in the inferior olive of adult mice through adeno-associated viral vector injections, and then study the olivary input to the cerebellum with light stimulation of olivary axons in the cerebellum. This presents an important advance in our ability to dissect cerebellar circuits, as previously electrical stimulation only allowed mixed activation of various fiber pathways. Most importantly, the deep cerebellar nuclei (DCN), which provide the final output from the cerebellum, receive excitatory input from olivary axons, but this functional significance of this pathway has never been isolated. In contrast, Purkinje cells in the cerebellar cortex receive olivary input in the form of climbing fibers, which have been extensively studied as each individual input is extremely strong and can easily be identified as a climbing fiber response. We will determine the effect of olivary input to the DCN using both brain slice recordings and recordings from animals with an intact cerebellar network. The slice recordings will allow us to determine the detailed synaptic properties of the olivary connection to DCN neurons for the first time. The recordings from intact animals will shed light on the impact of this connection in the intact network and whether the same olivary axons exciting a DCN neuron will also lead to a delayed inhibition of the same neuron via Purkinje cell climbing fiber activation. Overall our optogenetic studies address the actions of an important cerebellar input pathway, which hitherto could not be studied with classical techniques. We expect that significant excitatory effects on the DCN balancing inhibitory action of Purkinje cell input will be found. This knowledge will lay the basis for future studies examining the involvement of the olivary input pathway in cerebellar disease states in mouse mutants of ataxia and dystonia. PUBLIC HEALTH RELEVANCE: The cerebellum is the source of debilitating motor disorders, most notably cerebellar ataxias and some forms of dystonia. To better understand and treat these diseases we will study excitatory input to the deep cerebellar nuclei with optogenetic methods allowing a detailed analysis of this pathway for the first time. This analysis will help us to examine changes in cerebellar activity in mouse mutants causing cerebellar disease in future studies. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page