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。拟议的研究将解决神经营养因子在三叉神经(V)初级传入发育中的营养作用。我们的工作假设是神经营养因子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树突形成。此外，共培养的V轴突与V脑干细胞发生突触相互作用，这与在体内观察到的相似。这些数据促使我们提出以下3个假设；1. 胚胎小鼠V外周和脑干离子共培养，NGF产生轴突伸长，NT3产生轴突树枝化。为了验证这一点，我们将在外源添加NGF或NT3的情况下培养胚胎V外植体共培养和整体培养。在这些条件下，将量化树木砍伐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。轴突的生长模式也将在NGF或NT3增强（或适当的阻断剂）条件下的解离三叉细胞培养中进行研究。外植体共培养也将用于评估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.在可控启动子下，在脑干过表达NGF或NT3的转基因小鼠中，V神经节细胞将分别出现异常的轴突伸长和树突化特性。相反，在脸须中过度表达NGF或NT3的转基因小鼠将在脑干中发育正常的轴突形态。为了验证这一点，我们将在脑干或须垫中产生NGF或NT3可控过表达的转基因小鼠，通过使用CORE b中四环素调控的基因系统，在脑干或须垫中产生NGF或NT3可控过表达的转基因小鼠。在四环素存在的情况下，NGF或NT3将在特定的目标组织中在特定的发育时间过度表达。在这些转基因小鼠成年后，将通过轴突内记录和染色来揭示鉴定的V初级传入事件的形态。轴突生长模式将在CORE C. 3中量化。在转基因小鼠中，脑干细胞（小管）的地形聚集模式将不存在，这些小鼠表现出由脑干中NGF或NT3的控制过表达引起的异常的初级传入延伸和树枝化特性。这种模式异常会传递到丘脑样桶和皮质桶。为了验证这一点，将对上述NGF或NT3过表达的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。

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.