Uncovering the mechanism(s) responsible for a novel neurite co-orientation process.

本项目的主要目标是揭示神经外植体在组织培养中表现出共同定向神经突生长的机制。主要观察结果是，鸡胚神经组织的外植体，无论是交感神经节或感觉神经节，表现出不对称的生长与相邻外植体表现出相同方向的优先生长。已经排除了全局场效应（如重力）的可能性。不能排除其他机制，如扩散或基质梯度，但难以与现有数据相一致。一个有趣的替代方法是外植体建立电场，介导与附近外植体的通信。虽然在神经元组织内产生电场以及这种组织对外部场的响应性都已经很好地建立，但是为了协调同一组织内的生物过程而产生和响应这种场将是完全新颖的。有两个研究目标。首先是确定这种现象是否发生在其他物种（特别是哺乳动物和昆虫）的类似外植体中。第二个是研究电活动在介导共同取向的产物中的潜在作用。申请人将参与一项多学科研究计划，旨在利用四位不同科学家的专业知识，他们将在以下领域提供培训和监督：原代神经培养，形态测定分析，多电极阵列记录，活动成像和单细胞膜片钳记录。三个最初的8周培训轮换将是：1）在基思Crutcher和Curtis多布森的监督下使用主要神经组织培养方法，2）在Tim Brown的监督下使用多电极阵列电生理学，3）在Richard Baines的监督下使用单细胞电生理学和活动成像方法。完成旋转后，最初的实验将涉及建立鸡胚交感神经节的培养物，以复制最初的发现，如果在旋转过程中还没有完成的话。下一步将是确定在鸡外植体中观察到的现象是否也发生在小鼠感觉神经节和/或果蝇中枢神经元培养物中。这些实验将涉及：1）解剖组织，2）建立原代培养物，3）染色和分析所得的生长物。如果小鼠和/或果蝇外植体发生共同定向生长（从而证明普遍存在），这开辟了一个可能的未来调查路线，利用这两个额外的模型来探测潜在的机制，利用众多的遗传工具。如果小鼠/果蝇组织没有显示出与鸡组织相同的结果，重点将仍然是使用鸡组织来探测电场介导生长效应的可能性。这些实验将由Tim Brown监督，他目前使用多电极阵列来研究小鼠脑切片的活动。最初轮换期间的培训将使用现有方法，以确保申请人熟悉该方法。只有这样，该方法才适合于使用小鸡（和/或小鼠/苍蝇）外植体，第一个问题是是否存在电活动的证据，如果存在，是否存在任何协调或同步性的指示。如果没有发现这样的证据，将探索替代机制，如扩散化学梯度。如果涉及电活动，将在Richard Baines的指导下使用单细胞记录技术和/或全局活动成像（使用Ca 2+报告者）进行额外的研究，目的是揭示可能的细胞机制。

The primary goal of this project is to uncover the mechanism(s) by which neural explants exhibit co-oriented neurite growth in tissue culture. The primary observation is that explants of chick embryonic neural tissue, either sympathetic or sensory ganglia, show asymmetric outgrowth with neighbouring explants exhibiting preferential growth in the same direction. The possibility of a role for a global field effect, such as gravity, has been excluded. Alternative mechanisms, such as diffusible or substrate gradients, cannot be excluded but are difficult to reconcile with the available data. An intriguing alternative is that the explants establish electric fields that mediate communication with nearby explants. Although the generation of electrical fields within neuronal tissues, and the responsiveness of such tissues to external fields have both been well established, generation and response to such fields in order to coordinate biological processes within the same tissue would be entirely novel. There are two research aims. The first is to determine whether this phenomenon occurs in analogous explants from other species (specifically mammalian and insect). The second is to examine the potential role of electrical activity in mediating the co-oriented outgrowth. The applicant will engage in a multi-disciplinary research program designed to draw on the expertise of four different scientists who will provide training and oversight in the following areas: primary neural cultures, morphometric analysis, multi-electrode array recordings, activity-imaging and single cell patch-clamp recordings. The three initial 8-week training rotations will be: 1) primary neural tissue culture methods under the supervision of Keith Crutcher and Curtis Dobson, 2) the use of multi-electrode array electrophysiology under the supervision of Tim Brown and, 3) the use of single cell electrophysiological and activity-imaging methods supervised by Richard Baines. After completing the rotations, the initial experiments will involve establishing cultures of embryonic chick sympathetic ganglia to replicate the initial findings if not already accomplished during the rotation. The next step will be to determine whether the phenomenon observed with chick explants also occurs with mouse sensory ganglia and/or Drosophila central neuron cultures. These experiments will involve: 1) dissecting the tissue, 2) establishing primary cultures and 3) staining and analyzing the resulting outgrowth. If co-oriented outgrowth occurs with mouse and/or Drosophila explants (thereby demonstrating ubiquity), this opens up a possible future line of investigation to take advantage of the numerous genetic tools available using these two additional models to probe underlying mechanisms. If mouse/Drosophila tissue does not show the same results as the chick tissue, the focus will remain on using the chick tissue to probe the possibility that electric fields are mediating the growth effect. These experiments will be supervised by Tim Brown who currently uses multi-electrode arrays to study the activity of mouse brain slices. Training during the initial rotation will use the existing methods to ensure the applicant is familiar with the approach. Only then will the method be adapted to the use of the chick (and/or mouse/fly) explants with the first question being whether or not there is evidence of electrical activity and, if there is, whether there is any indication of co-ordination or synchronicity. If no such evidence is found, alternative mechanisms will be explored, such as diffusible chemical gradients. If electrical activity is implicated, additional studies using single cell recording techniques and/or global activity imaging (using Ca2+-reporters) under the direction of Richard Baines will be carried out with the goal of uncovering possible cellular mechanisms.