Ontogeny of CL- homeostasis in spherical bushy cells (SBC) on the anteroventral cochlear nucleus (AVCN): impact on GABAergic and glycinergic neurotransmission

前腹侧耳蜗核 (AVCN) 上球形丛状细胞 (SBC) CL-稳态的个体发育：对 GABA 能和甘氨酸能神经传递的影响

• 批准号: 5452597
• 负责人: Professor Dr. Ivan Milenkovic
• 金额: --
• 依托单位: Abteilung für Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5452597?language=en
• 关键词: Ontogeny; CL; homeostasis; spherical; bushy

Spherical bushy cells (SBC) of the mammalian cochlear nucleus transmit phaselocked activity of the auditory nerve in pathways that contribute to sound localization based on interaural time differences. However, the firing of SBC is determined by an interaction between excitatory (glutamatergic) and inhibitory inputs (GABAergic and glycinergic). Depending on the Cl- gradient across the membrane, activation of GABAA and glycine receptors can either depolarize neurons or, like in most mature neurons, hyperpolarize them. The aim of this project is to investigate the ontogeny of the Cl- regulation mechanisms and their outcome on the inhibitory neurotransmission in SBC. We will conduct three series of experiments: First, we intent to investigate the developmental pattern of the KCC2, a neuronal specific isoform of the K+-Cl- cotransporter which is, by extruding internal Cl-, critical for inhibitory GABA responses in mature neurons. Immunofluorescence and Western blot studies, during early postnatal development (before and after hearing onset), will examine the expression pattern of the KCC2 in SBC. Using brain slice preparations, gramicidin-perforated patch clamp recordings of SBC will scrutinize the driving force for Cl- by determining EGABA, EGly, and Vrest. Second, we will address the basis for excitatory GABAergic activity in SBC which we observed even after the hearing onset (P12). These experiments will explore the activity of the Cl¿accumulating Na+-K+-2Cl- cotransporter (NKCC1) which is responsible for excitatory GABAergic activity in diverse immature brain structures and in mature sensory neurons. Using loop diuretic drugs and assuming the differential activity pattern of both Clcotransporters throughout development, we intent to challenge Cl--accumulation early in development and Cl--extrusion in later stages which is, most likely, correlated with KCC2 activity. Third, Ca2+-imaging measurements around the hearing onset will address the GABA- and glycine-evoked [Ca2+]i responses since they, indirectly, provide evidence about the nature (depolarizing or hyperpolarizing) of the Cl- gradient and, directly, about the effects of inhibitory transmission on the Ca2+ signalling in SBC. If NKCC1 does not accompany the depolarizing action of GABA and glycine in SBC, our research will focus on another potential candidate which was recently postulated to render depolarizing Cl- gradient in auditory brainstem neurons, HCO3 -/Cl- exchanger AE3. Better understanding of the chloride regulation mechanisms in SBC will provide new insight into roles of GABA and glycine in coding acoustic signals at the first central synapse of the auditory pathway.

哺乳动物耳蜗核的球形丛状细胞（SBC）在基于耳间时间差的声音定位通路中传递听觉神经的锁相活动。然而，SBC的放电是由兴奋性（谷氨酸能）和抑制性输入（GABA能和甘氨酸能）之间的相互作用决定的。依赖于跨膜的Cl-梯度，GABAA和甘氨酸受体的激活可以使神经元去活化，或者像大多数成熟神经元一样使它们过度活化。本研究的目的是探讨SBC中Cl-调节机制的个体发生及其对抑制性神经传递的影响。我们将进行三个系列的实验：首先，我们打算调查的KCC 2，神经元特异性亚型的K+-Cl-的协同转运体，这是通过挤压内部Cl-，在成熟的神经元抑制GABA反应的关键的发展模式。免疫荧光和蛋白质印迹研究，在出生后的早期发展（听力发作之前和之后），将检查SBC中KCC 2的表达模式。使用脑切片制备，短杆菌肽穿孔膜片钳记录SBC将仔细检查Cl-的驱动力，通过确定EGABA，EGly，和Vrest。第二，我们将讨论SBC中兴奋性GABA能活动的基础，我们甚至在听力开始后（P12）也观察到这种活动。这些实验将探索Cl-积累Na+-K+-2Cl-协同转运蛋白（NKCC 1）的活性，NKCC 1负责不同未成熟脑结构和成熟感觉神经元中的兴奋性GABA能活性。使用袢利尿药物，并假设在整个发育过程中两种Cl转运蛋白的活性模式不同，我们打算挑战发育早期的Cl-积累和后期的Cl-排出，这很可能与KCC 2活性相关。第三，Ca 2+成像测量周围的听力发作将解决GABA和甘氨酸诱发的[Ca 2 +]i的反应，因为他们，间接地，提供证据的性质（去极化或超极化）的Cl-梯度，并直接，抑制性传输的影响SBC中的Ca 2+信号。如果NKCC 1不伴随SBC中GABA和甘氨酸的去极化作用，我们的研究将集中在另一个潜在的候选人，这是最近假设，使去极化Cl-梯度在听觉脑干神经元，HCO 3-/Cl-交换AE 3。更好地了解SBC中的氯调节机制将为GABA和甘氨酸在听觉通路第一中枢突触编码声信号中的作用提供新的见解。