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

哺乳动物耳蜗核的球形丛细胞（SBC）传递听神经相锁活动的途径有助于基于耳间时差的声音定位。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。根据膜上Cl-梯度的不同，GABAA和甘氨酸受体的激活既可以使神经元去极化，也可以像大多数成熟神经元一样使它们超极化。本项目旨在探讨Cl-调控机制的个体发生及其对SBC中抑制性神经传递的影响。我们将进行三个系列的实验：首先，我们打算研究KCC2的发育模式，KCC2是K+-Cl-共转运体的神经元特异性异构体，通过挤出内部Cl-，在成熟神经元中抑制GABA反应至关重要。免疫荧光和Western blot研究，在出生后早期发育期间（听力发作前后），将检测KCC2在SBC中的表达模式。使用脑切片制剂，革兰霉素穿孔膜片钳记录SBC将通过测定EGABA、EGly和Vrest来仔细检查Cl-的驱动力。其次，我们将解决SBC中兴奋性gaba能活性的基础，我们甚至在听力发作后观察到（P12）。这些实验将探索Cl-积累Na+- k +- 2cl -共转运体（NKCC1）的活性，NKCC1负责各种未成熟大脑结构和成熟感觉神经元的兴奋性gaba能活性。使用环利尿药物并假设两种clco转运蛋白在整个发育过程中的活性模式不同，我们打算挑战发育早期的Cl-积累和后期的Cl-挤压，这很可能与KCC2活性相关。第三，听力发作前后的Ca2+成像测量将解决GABA和甘氨酸诱发的[Ca2+]i反应，因为它们间接提供了Cl-梯度性质（去极化或超极化）的证据，并直接提供了SBC中抑制传递对Ca2+信号传导的影响。如果NKCC1在SBC中不伴随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物质上，HCO3 -/Cl-交换剂AE3，最近被假设在听觉脑干神经元中呈现去极化的Cl-梯度。更好地理解SBC中氯离子的调控机制，将为GABA和甘氨酸在听觉通路第一中枢突触编码声音信号中的作用提供新的见解。

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.