Plasticity of spinal L3 propriospinal neurons in urination recovery after thoracic SCI

摘要 膀胱功能恢复是脊髓损伤患者的当务之急，但到目前为止几乎没有有效的方法。 治疗。一个主要原因是对两者自发的椎管内机制的了解有限 脊髓损伤后的可塑性和治疗功能恢复。横断性脊髓损伤剥夺了椎管上的控制权 外道括约肌(EUS)，瘫痪其松弛功能，并导致尿潴留。部分 只有当腰3(L3)之间的神经回路发生时，EUS松弛和排尿才会自发恢复 腰椎6(L6)脊髓水平保持完好。电刺激可进一步改善排尿功能 在慢性脊髓损伤模型中刺激L3脊髓。然而，其背后的细胞机制尚不清楚。 在我们的初步研究中，我们应用了顺行跨单突触AAV1-GFP-Cre病毒，并发现 L3固有脊髓神经元群投射到L6背侧连合，并与 EUS相关中间神经元(INS)。我们将这些PSN命名为PSNsL3-L6 DCM。对这些的光遗传刺激 PSNsL3-L6 DCM诱发的EUS运动神经元的场电位(MNEUS)和EUS肌电(EMGEUS) 潜力。这些结果表明PSNsL3-L6 DCM与EUS之间存在功能联系。我们假设 PSNsL3-L6 DCM参与脊髓环路调节EUS松弛和调节自发性 完全性胸部脊髓损伤后EUS松弛功能的恢复。为了评估生理功能 对于PSNsL3-L6 DCM，我们将注射重组病毒转导这些神经元并表达通道视紫红质 用于光遗传刺激或hM4di用于神经功能的化学生成抑制。我们将评估是否 通过膀胱测压、EMGEUS和EUS对这些神经元的操作影响EUS松弛和排尿功能 L6的DCM和MNEUS的场势记录。我们还将结合顺行和逆行跨突触 病毒示踪和免疫染色鉴定PSNsL3-L6 DCM及其神经递质表型 突触后神经元。然后，我们将进一步探讨T8脊髓横断损伤是否会增强突触 PSNsL3-L6 DCM与其靶细胞之间的联系介导自发性功能恢复 尤斯。我们将使用与上面类似的方法来验证在此 脊髓损伤后4周，EUS松弛的脊髓反射自发恢复。此外， 慢性抑制PSNsL3-L6 DCM将在脊髓损伤后4周立即进行，以确定原因 神经可塑性在EUS松弛和排尿功能恢复中的作用这项研究将 是第一个探索PSNS在EUS控制完好和损伤脊髓中的作用的人，从而扩展了我们的 了解EUS控制的细胞机制。这将为一些有希望的 未来的治疗方法，如电刺激脊髓，作为恢复膀胱功能的一种手段。

Abstract Bladder function recovery is a top priority among the SCI patient population, yet so far there are few effective therapies. One major reason is the limited understanding of intraspinal mechanisms for both spontaneous plasticity and for therapeutic functional recovery after SCI. A transection SCI deprives supraspinal control of the external urethral sphincter (EUS) which paralyzes its relaxation function and leads to urine retention. Partial recovery of EUS relaxation and voiding occurs spontaneously only if the neural circuits between lumbar 3 (L3) and lumbar 6 (L6) spinal cord level remain intact. Voiding function can be further improved by electrical stimulation of the L3 spinal cord in a chronic SCI model. However, the cellular mechanism underneath is unclear. In our preliminary study, we applied an anterograde trans-monosynaptic AAV1-GFP-Cre virus and found that a population of L3 propriospinal neurons (PSNs) projected to the L6 dorsal commissure (DCM) and synapsed with EUS-related interneurons (INs). We named these PSNs as PSNsL3-L6 DCM. Optogenetic stimulation of these PSNsL3-L6 DCM induced field potential of EUS Motoneurons (MNEUS) as well as EUS electromyography (EMGEUS) potential. These results indicate a functional connection between PSNsL3-L6 DCM and the EUS. We hypothesize that PSNsL3-L6 DCM participate in a spinal circuit to modulate EUS relaxation and mediate the spontaneous recovery of the EUS relaxation function after complete thoracic SCI. To evaluate the physiological function of PSNsL3-L6 DCM, we will inject recombinant viruses to transduce those neurons and express channelrhodopsin for optogenetic stimulation or hM4di for chemogenetic inhibition of neuronal function. We will evaluate whether manipulation of these neurons affects EUS relaxation and voiding function by bladder cystometry, EMGEUS, and field potential recording of DCM and MNEUS at L6. We will also combine anterograde and retrograde transsynaptic viral tracing and immunostaining to characterize the neurotransmitter phenotype of these PSNsL3-L6 DCM and their postsynaptic neurons. We will then further explore whether a T8 transection SCI will enhance synaptic connections between PSNsL3-L6 DCM and their target cells to mediate the spontaneous functional recovery of the EUS. We will use similar methods as above to verify whether more synaptic connections are established in this circuit at 4 weeks after SCI when the spinal reflex for EUS relaxation spontaneously recovers. Furthermore, chronic inhibition of PSNsL3-L6 DCM will be made immediately after SCI for 4 weeks to determine the causal contribution of neural plasticity to the functional recovery of EUS relaxation and voiding function. This study will be the first to explore the role of PSNs in EUS control in both intact and injured spinal cords, thus extending our understanding of cellular mechanisms of EUS control. This will provide a fundamental basis for some promising future therapies such as electrical stimulation of the spinal cord as a means of recovering bladder function.