Abstract：
BACKGROUND: Study of synaptic integrity using conventional electrophysiology is a gold standard for quantitative assessment of neurodegeneration. Fluorescence assisted single-synapse long-term potentiation (FASS-LTP) provides a high throughput method to assess the synaptic integrity of neurotransmission within and between different brain regions as a measure of pharmacological efficacy in translational models.
METHODS: We adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker. This allowed for gradual, uniform thawing, effectively separating the synaptosomes. Our study demonstrates FASS-LTP on four brain regions at 6- and 12-month periods in the 3xTg-AD mouse model, treating sibling cohorts with VU0155069 (a small molecule inhibitor) or vehicle (0.9 % saline).
RESULTS: Our findings demonstrate the robust ability of the FASS-LTP technique to characterize the functional synaptic integrity maintained by disease-treatment therapies in multiple brain regions longitudinally using frozen brain tissue.
METHODS: By providing a detailed, user-friendly protocol for this well-known analysis and including a recovery step improved the ability to robustly replicate the FASS-LTP between different brain regions. This may be extrapolated to a translational use on human clinical samples to improve understanding of the therapeutic impact on synaptic performance related to glutamate neurotransmission.
CONCLUSIONS: FASS-LTP method offers a robust analysis of synaptosomes isolated from frozen tissue samples, demonstrating greater reproducibility in rodent and human synapses in physiological and pathological states.
METHODS: We adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker. This allowed for gradual, uniform thawing, effectively separating the synaptosomes. Our study demonstrates FASS-LTP on four brain regions at 6- and 12-month periods in the 3xTg-AD mouse model, treating sibling cohorts with VU0155069 (a small molecule inhibitor) or vehicle (0.9 % saline).
RESULTS: Our findings demonstrate the robust ability of the FASS-LTP technique to characterize the functional synaptic integrity maintained by disease-treatment therapies in multiple brain regions longitudinally using frozen brain tissue.
METHODS: By providing a detailed, user-friendly protocol for this well-known analysis and including a recovery step improved the ability to robustly replicate the FASS-LTP between different brain regions. This may be extrapolated to a translational use on human clinical samples to improve understanding of the therapeutic impact on synaptic performance related to glutamate neurotransmission.
CONCLUSIONS: FASS-LTP method offers a robust analysis of synaptosomes isolated from frozen tissue samples, demonstrating greater reproducibility in rodent and human synapses in physiological and pathological states.
摘要：
背景：使用常规电生理学研究突触完整性是定量评估神经变性的金标准。荧光辅助单突触长时程增强(FASS-LTP)提供了一种高通量方法来评估不同脑区域内和之间的神经传递的突触完整性,作为转化模型中药理学功效的量度。
方法：我们通过在冷冻样品解冻过程中增加一个步骤，使现有方法适应了我们的目的，通过额外的步骤将它们放置在室温下的摇杆上30分钟,在摇床上恒定混合解冻后立即解冻。这允许渐进的，均匀解冻，有效地分离突触体。我们的研究表明，在3xTg-AD小鼠模型中，FASS-LTP在6个月和12个月的四个脑区，用VU0155069(小分子抑制剂)或媒介物(0.9%盐水)治疗同胞队列。
结果：我们的研究结果表明，FASS-LTP技术具有强大的能力，可以使用冷冻脑组织纵向表征疾病治疗疗法在多个脑区维持的功能性突触完整性。
方法：通过提供详细的，该众所周知的分析的用户友好协议以及包括恢复步骤的协议提高了在不同大脑区域之间稳健复制FASS-LTP的能力。这可以外推到对人类临床样品的翻译使用,以提高对与谷氨酸神经传递相关的突触性能的治疗影响的理解。
结论：FASS-LTP方法提供了从冷冻组织样本中分离的突触体的可靠分析，在生理和病理状态下，在啮齿动物和人类突触中表现出更大的可重复性。
方法：我们通过在冷冻样品解冻过程中增加一个步骤，使现有方法适应了我们的目的，通过额外的步骤将它们放置在室温下的摇杆上30分钟,在摇床上恒定混合解冻后立即解冻。这允许渐进的，均匀解冻，有效地分离突触体。我们的研究表明，在3xTg-AD小鼠模型中，FASS-LTP在6个月和12个月的四个脑区，用VU0155069(小分子抑制剂)或媒介物(0.9%盐水)治疗同胞队列。
结果：我们的研究结果表明，FASS-LTP技术具有强大的能力，可以使用冷冻脑组织纵向表征疾病治疗疗法在多个脑区维持的功能性突触完整性。
方法：通过提供详细的，该众所周知的分析的用户友好协议以及包括恢复步骤的协议提高了在不同大脑区域之间稳健复制FASS-LTP的能力。这可以外推到对人类临床样品的翻译使用,以提高对与谷氨酸神经传递相关的突触性能的治疗影响的理解。
结论：FASS-LTP方法提供了从冷冻组织样本中分离的突触体的可靠分析，在生理和病理状态下，在啮齿动物和人类突触中表现出更大的可重复性。
