Electrophysiological studies of synapse formation by regenerating CST axons

描述(申请人提供)：脊髓损伤后的大部分功能缺陷是由纵向投射并连接大脑和脊髓的神经纤维破坏引起的。原则上，有两种可能的策略来重建功能回路来修复这种通信的丧失：未受损的神经纤维可以被刺激萌发侧枝轴突并建立代偿性连接，受损的轴突可以被刺激生长穿过病变与其原始靶点重新连接。最近在促进皮质脊髓束(CST)的萌发和再生方面取得了重大进展，CST是控制运动的主要途径。目前尚不清楚；然而，这些萌发和再生的CST轴突是否能够重新建立功能性突触连接。这项建议解决了第二个步骤，即确定萌发或再生的CST轴突是否可以与它们在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切除后其对侧脊髓内轴突的健壮萌发，并促进CST轴突在脊髓损伤中前所未有的再生。然而，发芽或再生的轴突是否可以进行功能性突触连接仍不清楚。小鼠CST轴突的一个主要靶点是位于脊髓C11-L2节段的Clarke柱神经元，与CST非常接近。我们建议利用体内电生理学方法来评估CST轴突与Clarke‘s柱神经元形成功能性突触的再生能力。在第一个目标中，我们将通过对PTEN缺失的小鼠进行单侧锥体切开术来中断另一个CST，从而诱导其中一个CST中的轴突发芽进入对侧脊髓。我们将测试发芽的CST轴突是否通过选择性刺激CST建立功能性突触连接，同时从Clarke的柱状神经元细胞内记录。因此，我们可以测试轴突萌芽是否能与脊髓中适当的突触目标形成功能性突触。在第二个目标中，CST将在T10通过完全脊髓挤压而损毁双侧。这个手术过程中断了所有通过挤压区域投射的轴突。在允许PTEN缺失小鼠的CST轴突通过损伤再生后，我们将在细胞内记录从尾部到挤压的Clarke柱神经元，同时在挤压上方的颈部水平刺激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轴突萌发和再生的一个重要功能方面，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皮质脊髓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。 公共卫生相关性：脊髓损伤会导致严重的功能丧失，因为连接大脑和脊髓的神经纤维在损伤部位被破坏。最近在促进皮质脊髓束(CST)重新生长方面取得了重大进展，CST是控制运动的主要途径，但CST神经纤维是否能与损伤下方适当的神经细胞重新建立功能连接尚不清楚。拟议的实验将使用小鼠脊髓的电子记录来确定重新生长的CST神经纤维是否可以与这些神经细胞形成功能连接，从而为设计促进脊髓损伤后功能恢复的治疗策略提供见解。

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.