NEUROTROPHIN CONTROL OF TRIGEMINAL AXON DEVELOPMENT

先前的研究已经揭示了神经营养因子在感觉神经元发育中的营养作用。拟议的研究将解决三叉神经（V）初级传入发展中的神经营养因子的营养作用。我们的工作假设是，神经营养因子调节感觉轴突的生长速度和模式在一个关键时期的发展。在大鼠V外周和脑干的扩大共培养中的初步研究表明，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，这与体内观察到的相似。这些数据提示了以下3个假设：1。胚胎小鼠V外周和脑干的离子共培养物，NGF产生轴突伸长，NT 3产生轴突分支。为了测试这一点，我们将在外源性添加的NGF或NT 3存在下培养胚胎V外植体共培养物和全胚。将在这些条件下定量树枝化反应中的轴突生长速率。将使用免疫组织化学标记物来确定NGF和NT 3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NT 3增强（或适当的阻断剂）条件下，在分离的三叉神经细胞培养物中研究轴突生长模式。外植体共培养也将用于评估NGF和NT 3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.在转基因小鼠中，在可控启动子下，在脑干中过表达NGF或NT 3，V神经节细胞将分别发生异常轴突伸长和树枝化特性。相反，在Mystheskerpad中过度表达NGF或NT 3的转基因小鼠将在脑干中发育正常的轴突形态。为了测试这一点，将产生在脑干或须垫中具有受控的NGF或NT 3过表达的转基因小鼠，由此，通过使用CORE B中的四环素调节的基因系统，将产生在脑干或须垫中具有受控的NGF或NT 3过表达的转基因小鼠。在通过饮食或注射给予四环素的情况下，在发育的特定时间内，NGF或NT 3将在特定靶组织中过表达。将通过轴突内记录和染色揭示这些成年转基因小鼠中鉴定的V初级传入的形态。轴突生长模式将在CORE C中定量。3.在转基因小鼠中将不存在脑干细胞（barbillettes）的局部聚集模式，所述转基因小鼠显示由脑干中NGF或NT 3的受控过表达诱导的异常初级传入延长和树枝化特性。这种模式异常将被传递到丘脑桶状体和皮质桶。为了测试这一点，将对在脑干中显示改变的初级传入结构的上述NGF或NT 3过表达小鼠进行组织化学处理，以揭示脑干、丘脑和大脑皮层中的潜在模式改变。在CORE C中进行晶须相关图案的图像分析。

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.