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神经轴突的生长速度和方式。大鼠V外周和脑干扩大共培养的初步研究表明，NGF和NTF3分别影响轴突的延长和脑干杆的形成。此外，共同培养的V轴突与V脑干细胞发生突触相互作用，这与体内看到的类似。这些数据提示以下3个假说：1.胚胎小鼠V外周和脑干的离子共培养，NGF产生轴突延长，NT3产生轴突树枝。为了测试这一点，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，树枝反应中的轴突生长速率将被量化。免疫组织化学标记物将用于确定NGF和NT3是否影响所有或特定V神经节细胞轴突的生长。在NGF或NT3增强(或适当的阻断剂)的条件下，也将研究分离的三叉神经细胞培养中轴突的生长模式。外植体共培养也将被用来评估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.在可控启动子的作用下，在脑干中过表达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延长和树枝形成特性。相反，在肌囊须垫中过度表达NGF或NT3的转基因小鼠将在脑干发育正常的轴突形态。为了验证这一点，利用四环素调控基因系统，在脑干或胡须垫中产生NGF或NT3受控过表达的转基因小鼠。在四环素存在的情况下，NGF或NT3将在发育特定的时间在特定的靶组织中过表达。这些转基因小鼠成年后的轴突内记录和染色将揭示已识别的V初级传入神经元的形态。轴突生长模式将在核心C中被量化。3.脑干细胞(桶)的地形聚集模式在转基因小鼠中将不存在，这些转基因小鼠表现出由脑干中NGF或NT3的控制过表达而引起的初级传入延长和树枝状特征的异常。这样的图形异常将传递到丘脑桶状核和大脑皮层桶。为了测试这一点，上面描述的NGF或NT3过度表达的小鼠显示出脑干初级传入结构的变化，将进行组织化学处理，以揭示脑干、丘脑和大脑皮层潜在的模式变化。与晶须有关的图案的图像分析将在C芯进行。

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.