Electrophysiological studies of synapse formation by regenerating CST axons

通过再生 CST 轴突形成突触的电生理学研究

• 批准号: 8234632
• 负责人: ERIC FRANK
• 金额: $ 26.29万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234632
• 关键词: Ablation; Action Potentials; Address; Adult; Amphibia; Axon; Brain; Cervical; Chest; Communication; Contralateral; Corticospinal Tracts; Electrons; Fiber; Genetic; Growth; Injury; Laboratories; Lesion; Location; Microscopic; Motor Neurons; Movement; Mus; Natural regeneration; Nerve Fibers; Neurons; Operative Surgical Procedures; PTEN gene; Pathway interactions; Population; Primates; Protocols documentation; Publishing; Pyramidal Tracts; Rattus; Recovery of Function; Research Personnel; Sensory; Site; Spinal; Spinal Cord; Spinal Cord Lesions; Spinal Injuries; Spinal cord injury; Synapses; Testing; Therapeutic; axonal sprouting; central nervous system injury; design; dorsal column; extracellular; in vivo; injured; innovation; insight; loss of function; repaired; research study; skills; synaptic function; synaptogenesis

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.

描述（由申请人提供）：脊髓损伤后的大多数功能缺陷是由纵向突出并互连大脑和脊髓的神经纤维破坏引起的。原则上，有两种可能的策略可以重建功能回路来修复这种通讯损失：可以刺激未受损的神经纤维发芽侧支轴突并建立补偿连接，并且可以刺激受伤的轴突在病变处生长与原来的目标重新建立联系。最近在促进皮质脊髓束（CST）的萌芽和再生方面取得了重大进展，皮质脊髓束是控制运动的主要途径。目前尚不清楚；然而，如果这些发芽和再生的 CST 轴突能够重新建立功能性突触连接。该提案解决了第二步问题，即确定发芽或再生的 CST 轴突是否可以与其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 的基因缺失会导致对侧 CST 消融后，其轴突在脊髓中对侧产生强劲的萌芽，并且还促进跨脊髓病变的 CST 轴突前所未有的再生。然而，发芽或再生的轴突是否可以形成功能性突触连接仍然未知。 小鼠 CST 轴突的主要目标是克拉克柱神经元，位于脊髓的 C11 - L2 段，靠近 CST。我们建议利用体内电生理学方法来评估重新生长的 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中断另一个 CST，从而诱导一个 CST 中的轴突发芽到对侧脊髓中。我们将通过选择性刺激 CST 同时从 Clarke 柱神经元进行细胞内记录来测试发芽的 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 将因 T10 处的脊髓完全挤压而受到双侧损伤。这种外科手术会中断穿过挤压区域的所有轴突。在 PTEN 缺失的小鼠中，让 CST 轴突通过损伤再生后，我们将在挤压物上方的颈部水平刺激 CST 的同时，从位于挤压物尾部的克拉克柱神经元进行细胞内记录。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。 总而言之，这些实验将使我们能够评估 CST 轴突的萌芽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。这些结果应该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）的再生方面取得了重大进展，皮质脊髓束是控制运动的主要途径，穿过受伤部位，但尚不清楚 CST 神经纤维是否可以与下方适当的神经细胞重新建立功能连接伤害。拟议的实验将使用小鼠脊髓的电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。