GDNF AND BDNF EFFECTS ON AGE ISOLATED DOPAMINE NEURONS

GDNF和BDNF是腹侧中脑多巴胺神经元发育、维持和衰老的两个重要因素。然而，无法研究GDNF或BDNF的完全零突变对衰老的影响，因为零突变的动物在出生后不久就会死亡。在0005项目中，我们建议使用器官型组织培养和移植技术来研究大鼠腹侧中脑多巴胺神经元的发育、维持和老化。 来自GDNF或BDNF基因敲除和杂合子动物的组织。因此，GDNF-/-和BDNF-/-以及它们的杂合子对应的胎儿腹侧中脑器官型组织培养将被用于研究生长因子对多巴胺神经纤维形成的影响，并与野生型和杂合子组织培养相比较(目标1)。为了检测GDNF或BDNF完全或部分敲除对中脑多巴胺神经元的老化效应，将胎儿腹侧中脑和外侧神经节隆起同时植入野生型小鼠的侧脑室，以建立由零突变组织组成的黑质纹状体微电路。然后在移植后3、12、18和24个月处死移植受者，以确定是否存在时间依赖性 在老化过程中，由于GDNF或BDNF零突变，移植组织中发生有害的变化。将从宿主动物身上获取脑脊液样本，以确定环境对移植组织的潜在影响。在所有移植类型的一组中，在移植前将GDNF和BDNF添加到孵化浴中，以确定补充适当的营养因子是否可以逆转GDNF或BDNF缺失的潜在影响(目标2)。将通过器官组织培养(用于发育研究)和移植(用于衰老研究)来研究GDNF或BDNF耗竭和出生前暴露于脂多糖(LPS)导致的早期神经炎症的联合影响。最后，将研究GDNF或BDNF缺失突变的黑质纹状体移植物暴露于环境毒素(甲基苯丙胺)的情况(目标3)。项目0005的总体目标是研究在缺乏GDNF或BDNF的情况下，结合神经炎症和环境毒素的影响，中脑多巴胺神经元发育和老化的命运。此外，项目0005为其他项目提供了两个水平的对照研究：A)可在移植物或组织培养中研究零突变动物以确定完全耗尽BDNF或GDNF的影响，以及B)我们将能够确定在项目0001-0004中观察到的影响是否源于体内的大脑 由于我们将在颅内移植和组织培养中分离出有问题的大脑区域，因此，我们将对特定的、改变或全身影响的脑区进行研究。

GDNF and BDNF are two important factors for development, maintenance, and aging of ventral mesencephalic dopamine neurons. It is, however, not possible to study aging effects of complete null mutations of either GDNF or BDNF, since null mutated animals die shortly after birth. In project 0005 we propose to use organotypic tissue culture and grafting techniques to study the development, maintenance, and aging of ventral mesencephalic dopamine neurons in tissue derived from GDNF or BDNF knockout and heterozygous animals. Thus, organotypic tissue cultures of fetal ventral mesencephalon from GDNF-/- and BDNF-/- as well as their heterozygous counterparts will be utilized to study growth factor effects on dopamine nerve fiber formation in comparison with wildtype and heterozygous tissue cultures (Aim 1). To examine aging effects of GDNF or BDNF complete or partial knockout on midbrain dopamine neurons in isolation, fetal ventral mesencephalon and lateral ganglionic eminence will be implanted simultaneously into the lateral ventricle of wildtype mice, so that a nigrostriatal microcircuit will be created of null mutated tissue. Recipients of transplants will then be sacrificed at 3, 12, 18, and 24 months post-grafting, to determine if there are time-dependent detrimental alterations occurring in transplanted tissues during aging as a result of the GDNF or BDNF null mutation. CSF samples will be obtained from the host animals to determine potential influence of the environment on grafted tissues. GDNF and BDNF will be added to the incubation bath prior to grafting in one group of all transplant types to determine if supplementation with the appropriate trophic factors may reverse potential effects of GDNF or BDNF deletion (Aim 2). Combined effects of GDNF or BDNF depletion and early neuroinflammation by prenatal exposure to lipopolysaccharide (LPS) will be studied using organotypic tissue culture (for development studies) and grafting (for aging studies). Finally, exposure to an environmental toxin (methamphetamine) in GDNF or BDNF null mutated nigrostriatal cografts will be studied (Aim 3). The overall goal with Project 0005 is to examine the fate of midbrain dopamine neurons developed and aged in the absence of GDNF or BDNF combined with effects of neuroinflammation and environmental toxins. Furthermore, Project 0005 provides two levels of control studies for the other projects: A) null mutated animals may be studied in grafts or tissue culture to determine the effects of a complete depletion of BDNF or GDNF, and B) we will be able to determine if the effects observed in Project 0001-0004 in vivo are due to intrinsic, brain specific, alterations or to systemic effects, since we will isolate the brain regions in question in intracranial transplants and tissue cultures.