Inducible neuronal inactivation in mice

描述（由申请人提供）：为了更好地理解中枢肾上腺素能信号传导的作用及其与神经精神疾病（如抑郁症）的关系，我们提出在小鼠中建立一种系统，用于诱导遗传定义的神经元失活。 虽然普遍适用，该系统将被应用于肾上腺素能和多巴胺能神经元的研究。 这种技术将提供一种有效的方法来确定这些神经元在体内的功能，无论是在发育过程中还是在成人中。 我们建议通过蛋白水解裂解突触囊泡与质膜融合所必需的蛋白质（SNARE）来抑制神经递质的释放。 我们将通过开发转基因小鼠品系来实现这一目标，这些小鼠可以表达具有蛋白水解活性的C。 破伤风（TeNT）或C. 肉毒杆菌（BoNT/E）神经毒素。 小鼠密码子优化的神经毒素转基因的表达将通过使用对反式激活因子的存在敏感的诱导型启动子与小分子诱导剂药物的组合来调节。 细胞特异性表达将通过将密码子优化的反式激活因子基因和哺乳动物内部核糖体进入位点靶向插入到限定感兴趣的神经元的基因的3 '-非翻译区来实现。 这种方法应该允许反式激活因子的真实表达而不破坏内源基因的表达。 在给予诱导剂药物后，神经元将被灭活。 该系统的优势可能是失活神经元的特异性（由于靶向方案）、失活的完整性（由于神经毒素的效力）和暂时控制的失活的稳定性（由于神经毒素作用的半衰期）。 我们提出的技术将适用于任何一组可以遗传定义的神经元。 我们将在多巴胺（DA）神经元中测试该系统，因为它们的失活被预测会导致运动功能减退和食欲减退的帕金森病表型。 DA释放的阻断将通过体内微透析和体外循环伏安法来评估。 该系统也将应用于肾上腺素能神经元的研究，使用类似的技术来记录神经递质释放的诱导性和可逆性失活。 该方法将允许在这些神经元失活之前和之后对动物进行研究，提供有价值的内部对照。 这项技术将补充用于研究相同神经元的其他技术，例如它们的遗传消融或从这些神经元中遗传消除单个神经递质。 这种技术的使用将为帕金森氏症或抑郁症等疾病提供一个模型，其中神经元群体被假设为功能障碍。 总的来说，这项技术的发展应该为解剖大脑如何运作提供了一个强大的工具。

DESCRIPTION (provided by applicant): In our desire to better understand the roles of central adrenergic signaling and their relation to neuropsychiatric conditions such as depression, we propose to create a system in mice for the inducible inactivation of genetically defined neurons. While generally applicable, this system will be applied to the study of adrenergic and dopaminergic neurons. Such a technique will provide a potent method for determining the functions of these neurons in vivo, either during development or in the adult. We propose to inactivate neurotransmitter release through proteolytic cleavage of one of the proteins (SNAREs) essential for synaptic vesicle fusion with the plasma membrane. We will achieve this by developing transgenic lines of mice that can express the proteolytically active light chain of either a C. tetani (TeNT) or C. botulinum (BoNT/E) neurotoxin. Expression of the mouse codon-optimized neurotoxin transgenes will be regulated by the use of an inducible promoter sensitive to the presence of a transactivation factor in combination with a small molecule inducer drug. Cell-specific expression will be achieved by targeting insertion of the codon-optimized transactivator gene and a mammalian internal ribosome entry site to the 3'-untranslated region of a gene that defines the neurons of interest. This approach should permit true expression of the transactivator without disrupting expression of the endogenous gene. Neurons would be inactivated following administration of the inducer drug. The strengths of this system are likely to be the specificity of the neurons inactivated (due to the targeting scheme), the completeness of inactivation (due to the potency of the neurotoxin), and the stability of the temporally controlled inactivation (due to the half-life of the neurotoxin's effects). Our proposed technique will be applicable to any set of neurons that can be genetically defined. We will test this system in dopamine (DA) neurons because their inactivation is predicted to result in the Parkinsonian phenotypes of hypomotility and hypophagia. Block of DA release will be assessed by microdialysis in vivo and cyclic voltammetry in vitro. The system will also be applied to the study of adrenergic neurons, using similar techniques to document inducible and reversible inactivation of neurotransmitter release. The approach will permit the study of animals before and after inactivation of these neurons, providing a valuable internal control. This technique will complement other techniques used to study the same neurons, such as their genetic ablation or the genetic elimination of a single neurotransmitter from those neurons. Use of this technique will provide a model for diseases such as Parkinson's or depression in which a neuronal population is hypothesized to become dysfunctional. In general, the development of this technique should provide a powerful tool for the dissection of how the brain operates.