Transporter-dependent regulation of glycine neurotransmission in the respiratory network: the role of GlyT1 (SLC6A9) GlyT2 (SLC6A5) and Asc-1 (SLC7A10)

呼吸是由延髓和脑桥的神经元网络控制的。虽然主要的节律发生器被证明是谷氨酸能的，但甘氨酸依赖的神经传递发挥着重要的调节功能，对呼吸网络的协调活动至关重要。细胞外甘氨酸浓度的调节是通过高亲和力、大容量的甘氨酸转运蛋白来实现的，该转运蛋白介导甘氨酸穿过突触前终末或突触周围的神经胶质细胞的膜。尽管大多数甘氨酸依赖突触的细胞都表达不止一种对甘氨酸具有高亲和力的转运蛋白，但它们之间的功能相互作用仍然知之甚少。在这个项目中，我们计划研究甘氨酸的高亲和力转运体，即主要由星形胶质细胞表达的甘氨酸转运体1(GlyT1；Slc6A9)和丙氨酸-丝氨酸-半胱氨酸-1转运体(ASC-1；Slc7A10)与神经元表达的GlyT2(Slc6a5)如何相互作用来调节突触的动态平衡，以及它们在发育过程中的作用如何变化。因此，我们不仅将加深对转运蛋白本身功能的理解，还将扩展我们的知识，即甘氨酸依赖的神经传递如何参与呼吸活动的调节。此外，它可能为某些药物(如全身麻醉药)如何发挥呼吸抑制作用的机制提供新的见解。我们正计划通过一种跨学科的方法来解决这个问题，使用转基因小鼠系，结合基于病毒的技术和RNAi方法来操纵不同转运蛋白的细胞表达。这将与生化和生理学方法相结合，以便能够在原位和体内分析小鼠的呼吸活动。综上所述，这将有助于我们描绘出各个转运蛋白在抑制性突触中的确切作用，并详细描述这些转运蛋白如何在功能上相互作用。这将包括描述各自的转运体对抑制性甘氨酸能突触甘氨酸周转的影响。因此，这个项目将加强我们对转运蛋白功能如何与呼吸神经元突触传递的改变和呼吸行为的相关性的理解。

Breathing is controlled by a neuronal network in the medulla and pons. Although the principal rhythm generator was shown to be glutamatergic, glycine-dependent neurotransmission exerts important modulatory functions and is critical for the coordinated activity of the respiratory network. The regulation of the extracellular glycine concentration is achieved by high affinity large capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminal or glial cells surrounding the synapse. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their functional interaction is only poorly understood. In this project, we now plan to investigated, how high affinity transporters for glycine, i.e. the predominantly astroglial expressed glycine transporter 1 (GlyT1; Slc6A9) and alanine-serine-cysteine-1 transporter (Asc-1; Slc7A10) and the neuronal expressed GlyT2 (Slc6a5) interact to regulate synapse homeostasis and how their contributions change during development. Thereby, we will not only foster our understand on the transporter functions per se, but will also extend our knowledge how glycine-dependent neurotransmission is involved in the regulation of the respiratory activity. In addition, it might provide novel insights on the mechanism how certain drugs like general anesthetics exert respiratory depressive effects. We are planning to tackle this topic by an interdisciplinary approach using genetically-modified mouse lines together with virus-based techniques and RNAi methods to manipulate the cellular expression of the different transporters. This will be combined with biochemical and physiological methods to allow the analysis of the respiratory activity in mice in-situ as well as in-vivo. Taken together this will help us to delineate the precise roles of the respective transporters at inhibitory synapses and describe in detail how these transporters interact functionally. This will include the description of the impact of respective transporters on glycine turnover at inhibitory glycinergic synapses. Thereby this project will enhance our understanding how transporter function correlates with alteration in synaptic transmission in respiratory neurons and with the breathing behavior.