Inducible neuronal inactivation in mice

描述（由申请人提供）：我们希望更好地理解中枢肾上腺素能信号的作用及其与神经精神疾病（如抑郁症）的关系，我们建议在小鼠中创建一个系统，用于诱导基因定义的神经元失活。虽然一般适用，但该系统将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。我们建议通过蛋白质水解裂解突触囊泡与质膜融合所必需的一种蛋白质（SNAREs）来灭活神经递质释放。我们将通过开发能够表达C.破伤风杆菌（TeNT）或C.肉毒杆菌（BoNT/E）神经毒素蛋白水解活性轻链的转基因小鼠来实现这一目标。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激活因子存在敏感的诱导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激活子基因和哺乳动物内部核糖体进入位点插入到定义感兴趣神经元的基因的3'-未翻译区域来实现。这种方法应该允许在不破坏内源基因表达的情况下真正表达反激活子。在使用诱导剂药物后，神经元会失活。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），以及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被基因定义的神经元。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帕金森病的运动能力低下和吞咽不足表型。将通过体内微透析和体外循环伏安法评估DA释放阻滞。该系统还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可诱导和可逆的神经递质释放失活。该方法将允许研究这些神经元失活前后的动物，提供有价值的内部控制。这项技术将补充用于研究相同神经元的其他技术，例如它们的基因消融或从这些神经元中遗传消除单个神经递质。这项技术的使用将为帕金森病或抑郁症等疾病提供一个模型，在这些疾病中，神经元群被假设为功能失调。总的来说，这项技术的发展应该为解剖大脑的运作方式提供一个强有力的工具。

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.