Electrophysiological studies of synapse formation by regenerating CST axons

描述（由申请人提供）：脊髓损伤后的大多数功能缺损是由于纵向投射并连接大脑和脊髓的神经纤维断裂引起的。原则上，有两种可能的策略来重建功能回路，以修复这种通信损失：可以刺激未受损的神经纤维长出侧支轴突并建立代偿连接，并且可以刺激受伤的轴突在病变中生长以重新连接其原始目标。皮质脊髓束（CST）是控制运动的主要途径，近年来在促进其发芽和再生方面取得了重大进展。这是未知的；然而，如果这些萌发和再生的CST轴突可以重建功能性突触连接。这个建议解决了第二步，确定发芽或再生的CST轴突是否可以与脊髓中的正常目标神经元进行功能性突触接触。最近的研究表明，CST神经元中PTEN的基因缺失导致对侧CST消融后脊髓中对侧轴突的强劲发芽，并促进脊髓病变中CST轴突的前所未有的再生。然而，发芽或再生的轴突是否能形成功能性的突触连接尚不清楚。小鼠CST轴突的主要目标是克拉克柱神经元，位于脊髓C11 - L2节段，靠近CST。我们建议利用体内电生理方法来评估再生CST轴突与克拉克柱神经元形成功能性突触的能力。在第一个目标中，我们将通过pten缺失小鼠的单侧锥体切开术打断另一个CST，从而诱导一个CST的轴突长出对侧脊髓。我们将通过选择性地刺激CST，同时记录克拉克柱神经元的细胞内，来测试发芽的CST轴突是否建立功能性突触连接。因此，我们可以测试轴突芽是否可以在脊髓中形成具有适当突触目标的功能性突触。在第二个目标中，CST将通过T10完全性脊髓挤压而双侧受损。这种外科手术阻断所有通过挤压区投射的轴突。在pten缺失小鼠的损伤中允许CST轴突再生后，我们将记录细胞内Clarke's柱神经元在压伤处的尾侧，同时刺激压伤处以上颈椎水平的CST。这些实验将测试通过病变再生的CST轴突是否能够在病变下方形成功能性突触连接。综上所述，这些实验将使我们能够评估CST轴突发芽和再生的重要功能方面，即它们形成功能性突触连接的能力。这些结果将为设计重建脊髓皮质连接和促进脊髓损伤后功能恢复的治疗策略提供直接的见解。

DESCRIPTION (provided by applicant): Most functional deficits after spinal cord injury are caused by the disruption of nerve fibers that project longitudinally and interconnect the brain and spinal cord. In principle, there are two possible strategies for re-building functional circuits to repair this loss of communication: nerve fibers that were not damaged can be stimulated to sprout collateral axons and build compensatory connections, and injured axons can be stimulated to grow across the lesion to reconnect with their original targets. Major progress has been made recently in promoting sprouting and regeneration of the corticospinal tract (CST), a major pathway for controlling movement. It is not known; however, if these sprouted and regenerated CST axons can re- establish functional synaptic connections. This proposal addresses that second step, determining if sprouting or regenerating CST axons can make functional synaptic contacts with their normal target neurons in the spinal cord. Recent studies have shown that genetic deletion of PTEN in CST neurons results in robust contralateral sprouting of their axons in the spinal cord following ablation of the contralateral CST and also promotes unprecedented regeneration of CST axons across spinal cord lesions. It remains unknown, however, if sprouted or regenerated axons can make functional synaptic connections. A major target of CST axons in mice are Clarke's column neurons, located in the C11 - L2 segments of the spinal cord, in close proximity to the CST. We propose to utilize in vivo electrophysiological approaches to assess the ability of re-growing CST axons form functional synapses with Clarke's column neurons. In the first aim, we will induce the axons in one CST to sprout into the contralateral spinal cord by interrupting the other CST via a unilateral pyramidotomy in PTEN-deleted mice. We will test if sprouted CST axons establish functional synaptic connections by selectively stimulating the CST while recording intracellularly from Clarke's column neurons. We can thus test if axonal sprouts can form functional synapses with appropriate synaptic targets in the spinal cord. In the second aim, the CST will be lesioned bilaterally by a complete spinal cord crush at T10. This surgical procedure interrupts all axons that project through the crush region. After allowing CST axons to regenerate through the lesion in PTEN-deleted mice, we will record intracellularly from Clarke's column neurons just caudal to the crush while stimulating the CST at cervical levels above the crush. These experiments will test if CST axons regenerating through the lesion are able to form functional synaptic connections below the lesion. Taken together, these experiments will allow us to assess an important functional aspect of sprouting and regenerating CST axons, namely their ability to form functional synaptic connections. These results should provide direct insights into designing therapeutic strategies for re-establishing corticospinal connections and promoting functional recovery after spinal cord injuries. PUBLIC HEALTH RELEVANCE: Spinal cord injuries cause a major loss of function because nerve fibers that connect the brain and spinal cord are disrupted at the site of injury. Major progress has been made recently in promoting re-growth of the corticospinal tract (CST), a major pathway for controlling movement, across the injured site, but it is unknown if CST nerve fibers can re-establish functional connections with appropriate nerve cells below the injury. The proposed experiments will use electrical recordings from mouse spinal cords to determine if re- growing CST nerve fibers can form functional connections with these nerve cells, thus providing insights into designing therapeutic strategies for promoting functional recovery after spinal cord injuries.