NEUROTROPHIN CONTROL OF TRIGEMINAL AXON DEVELOPMENT

神经营养因子对三叉神经轴突发育的控制

• 批准号: 6584614
• 负责人: MARK F JACQUIN
• 金额: $ 5.31万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584614
• 关键词: axon; cell cell interaction; developmental neurobiology; embryo /fetus tissue /cell culture; genetically modified animals; immunocytochemistry; laboratory mouse; mixed tissue /cell culture; neurogenesis; neurotrophic factors; trigeminal nerve

Prior studies have revealed the trophic actions of neurotrophins in sensory neuron development. Proposed studies will address the trophic actions of neurotrophins in trigeminal (V) primary afferent development. Our working hypothesis is that neurotrophins regulatory sensory axon growth rate and mode during a critical period in development. Preliminary studies in expand co-cultures of the rat V periphery and brainstem show that NGF and NTF3 influence over axon elongation and brainstem arbor formation, respectively. Moreover, co-cultured V axons develop synaptic interactions with V brainstem cells that are similar to those seen in vivo. These data prompt the following 3 hypotheses; 1. Ion co-cultures of the V periphery and brainstem from embryonic mice, NGF produces axon elongation and NT3 produces axon arborization. TO test this, we will grow embryonic V explant co-cultures and wholemounts in the presence of exogenously added NGF or NT3. Axon growth rates in arborization responses will be quantified under these conditions. Immunohistochemical markers will be used to determine whether NGF and NT3 affect growth of all or specific V ganglion cell axons. Axon growth patterns will also be studied in dissociated trigeminal cell cultures under conditions of NGF or NT3 augmentation (or appropriate blocking agents). Explant co-cultures will also be used to assess whether NGF and NT3 promote synaptic interactions between V primary afferent and brainstem neurons. 2.In transgenic mice that over-express NGF or NT3 in the brainstem under a controllable promoter, V ganglion cells will develop aberrant axon elongation and arborization properties, respectively. Conversely, transgenic mice that over-express NGF or NT3 in the mysacial whiskerpad will develop normal axon morphologies in the brain stem. To test this, transgenic mice will be generated with controlled over- expression of NGF or NT3 in the brainstem or whiskerpad, by this, transgenic mice will be generated with controlled over-expression of NGF or NT3 in the brainstem or whiskerpad, by using the tetracycline- regulated gene system in CORE B. In the presence of tetracycline administered by diet or injection, NGF or NT3 will be over-expressed in particular target tissues during specified times in development. The morphology of identified V primary afferents will be revealed by intra- axonal recording and staining in these transgenic mice in adulthood. Axonal growth patterns will be quantified in CORE C. 3. Topographic aggregation patterns of brainstem cell (barrelettes) will be absent in transgenic mice that display aberrant primary afferent elongation and arborization properties induced by controlled over- expression of NGF or NT3 in the brainstem. Such patterning abnormalities will be transmitted to the thalamic barreloids and cortical barrels. To test this, above-described NGF or NT3 over-expressing mice that display altered primary afferent structure in the brainstem will be histochemically processed to reveal potential patterning alterations in the brainstem, thalamus and cerebral cortex. Image analysis of whisker- related patterns will be carried out in CORE C.

先前的研究已经揭示了神经营养素在感觉神经元发育中的营养作用。拟议的研究将探讨神经营养素在三叉神经 (V) 初级传入神经发育中的营养作用。我们的工作假设是，神经营养素在发育的关键时期调节感觉轴突的生长速度和模式。对大鼠 V 外周和脑干扩大共培养的初步研究表明，NGF 和 NTF3 分别影响轴突伸长和脑干乔木形成。此外，共培养的 V 轴突与 V 脑干细胞形成突触相互作用，类似于体内观察到的情况。这些数据提出了以下 3 个假设； 1.胚胎小鼠V外周和脑干的离子共培养，NGF产生轴突伸长，NT3产生轴突树枝化。为了测试这一点，我们将在外源添加的 NGF 或 NT3 存在的情况下培养胚胎 V 外植体共培养物和整体。在这些条件下，将量化树枝化反应中的轴突生长速率。免疫组织化学标记将用于确定 NGF 和 NT3 是否影响所有或特定 V 神经节细胞轴突的生长。还将在 NGF 或 NT3 增强（或适当的封闭剂）条件下在离解的三叉神经细胞培养物中研究轴突生长模式。外植体共培养物还将用于评估 NGF 和 NT3 是否促进 V 初级传入神经元和脑干神经元之间的突触相互作用。 2.在可控启动子作用下脑干中过度表达NGF或NT3的转基因小鼠中，V神经节细胞将分别产生异常的轴突伸长和树枝化特性。相反，在肌须垫中过度表达 NGF 或 NT3 的转基因小鼠将在脑干中发育出正常的轴突形态。为了测试这一点，将产生在脑干或须垫中受控地过度表达NGF或NT3的转基因小鼠，通过使用CORE B中的四环素调节基因系统，将产生在脑干或须垫中受控地过度表达NGF或NT3的转基因小鼠。在通过饮食或注射给予四环素的情况下，NGF或NT3 在特定的发育时期，将在特定的靶组织中过度表达。所鉴定的 V 初级传入神经的形态将通过这些成年转基因小鼠的轴突内记录和染色来揭示。轴突生长模式将在 CORE C 中量化。 3. 转基因小鼠中将不存在脑干细胞（桶）的拓扑聚集模式，这些小鼠表现出由脑干中 NGF 或 NT3 受控过度表达诱导的异常初级传入伸长和树枝化特性。这种模式异常将传递到丘脑桶状体和皮质桶。为了测试这一点，将对上述脑干中初级传入结构改变的 NGF 或 NT3 过度表达小鼠进行组织化学处理，以揭示脑干、丘脑和大脑皮层中潜在的模式改变。晶须相关图案的图像分析将在 CORE C 中进行。