内源性神经活动调节螺旋神经元在发育早期的突触精细化

Human being can sense, perceive, and respond to complex sounds in surrounding environment via a precisely-organized auditory circuit. In the cochlea of the inner ear, sounds waves are initially converted into electrical signals by specialized sensory receptor located in the inner hair cells (HCs), which are then transmitted to the central nervous system by the primary sensory neurons━spiral ganglion neurons (SGNs). SGNs convey the intensity, frequency and phase of sound waves into the brain by way of particular firing patterns, which are ultimately deciphered into the sound loudness, pitch and localization by the brain. Therefore, SGNs must develop accurate and elaborate synapses that connect hair cells in the cochlea with the neurons in the brainstem at the stage of early postnatal age. However, whether the refined synaptic formation and sequential maturation of SGNs are determined by gene expression or activity remains elusive. Our preliminary data indicate that the afferent fibers of type-I spiral ganglion neurons have undergone a dramatic re-organization and pruning during early postnatal development. Based on these results, we will investigate the following questions using whole-cell electrophysiological recordings, genetics, imaging and cell biology: (1) whether the dynamic synaptic refinement of SGNs correlates with their intrinsic membrane properties, firing patterns, synaptic transmission during the neonatal development; (2) whether sensory depletion ( deafness mice) can alter synaptic pruning and their auditory circuits assembly; (3) whether there is a critical period for a precise auditory circuit assembly using reversible gene expression in deafness mice through tamoxifen induction at different developing stages; (4) whether activity can change synaptic refinement and target selection during early postnatal development by viral expression of bacterial sodium channels (NaChBac) or a non-inwardly rectifying variant of the Kir2.1 (ESKir2.1). Results from this study will provide new insights into how spiral ganglion neurons undergo synaptic pruning and form elaborate circuit assembly in the mammalian cochlea, and shed some light on the best time window for treating hearing disorders.

人类获取声音信息依赖于完整、精细的听觉环路的形成。首先，耳蜗中的毛细胞把声音信息转化为电信号传递到螺旋神经元；然后，螺旋神经元通过特定放电模式和突触传递把声音的频率、强度和位置信息传递到大脑，并且被准确的解读。但是，螺旋神经元在发育过程中的成熟和突触精细化是由内部因素（基因）还是外部因素（活动）决定的还不清楚。我们的预实验结果表明，螺旋神经元在发育早期经历一个明显突触修剪过程；在此基础上，本课题拟利用膜片钳技术，结合基因手段、成像技术、细胞分子生物学方法，研究：（1）螺旋神经元在出生后早期突触精细化的动态过程和其膜特性、放电模式、突触形成相关性；（2）外部活动剥夺（耳聋）对螺旋神经元突触修剪以及神经环路形成的影响；（3）利用Tamoxifen在不同发育时期恢复耳聋小鼠缺失基因的表达，寻找发育关键时期；（4）改变神经元兴奋性对螺旋神经元突触修剪和靶向选择的调控，进一步阐明突触发生的内在机制。

在听力获得之前，听觉环路的自发性电活动在发育早期短暂存在，并且起源于耳蜗，具有特定的放电模式 (pattern of spontaneous activity) ，被认为指导了这种听觉系统精细环路的形成。然而，螺旋神经元在这种特定模式自发性电活动的起源中扮演一个什么角色，以及特定模式自发性电活动在发育中的动态变化还不清楚。通过膜片钳全细胞记录、成像技术结合三维重构手段，我们发现：（1）螺旋神经元的自发性电活动在发育早期 (postnatal, P3−7) 以簇状方式 (burst firing) 进行，并具有明显的区域性差异 (P6−7) ；然而在听力快开始时 (P9−10) ，簇状放电模式消失；（2）螺旋神经元的树突分支修剪略早于簇状放电1−2天，但是与自发性电活动具有同样的发育变化模式；（3）通过显性负效突变体 (dominant negative) 阻断螺旋神经元的小电导钙激活的钾通道 (small conductance calcium-activated potassium channels, SK) 或者超极化激活环核苷酸门控通道 (hyperpolarization-activated cyclic nucleotide-gated channels, HCN) 干扰早期的簇状放电模式会导致小鼠听力的丧失。这些结果揭示早期模式化的自发性电活动决定了发育中神经环路的正确连接和精细化；（4）进一步利用单细胞提取技术，通过转录子测序、分析，我们发现干扰螺旋神经元特定放电模式下调了线粒体功能相关和突触生长相关蛋白的mRNA表达，表明它们可能参与了发育早期的特定模式放电在听觉环路形成中的作用；和（5）shRNA敲减线粒体相关基因Them5导致于自发性电活动阻断相似的突触修剪异常和耳聋表型。. 总之，本课题深入研究了发育早期特定模式放电在听觉环路形成中的作用以及发现了一些特定模式自发性放电激活的基因。本课题的研究成果给特定模式自发性放电指导了听觉环路的发育提供重要的理论依据，在听觉系统发育中具有重要的生物学意义。