Role of Calcineurin Signaling in Membrane Potential-Regulated Maturation of Cerebellar Granule Cells
Role of Calcineurin Signaling in Membrane Potential-Regulated Maturation of Cerebellar Granule Cells
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DOI：
10.1523/jneurosci.5932-08.2009
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发表时间：
2009-03-04
影响因子：
5.3
通讯作者：
Nakanishi, Shigetada
中科院分区：
文献类型：
--
作者：
Okazawa, Makoto;Abe, Haruka;Nakanishi, Shigetada
在出生后早期，小脑颗粒细胞增殖、分化、迁移，最后与苔藓纤维形成精细的突触连接。在此期间，未成熟颗粒细胞的静息膜电位相对去极化，但在成熟细胞中则变得超极化。进行这项研究的目的是检查膜电位的这种变化及其下游信号传导机制在颗粒细胞发育和成熟中的作用。实验旨在通过 DNA 转染将器官型小脑培养物与颗粒细胞中 EGFP（增强型绿色荧光蛋白）的特异性表达相结合，精确表征颗粒细胞发育的个体发育过程。使用形态学、电生理学和免疫组织化学的多种方法证明，颗粒细胞在器官型培养物中的生理 KCl 浓度下以时间调节的方式发育和成熟。我们通过用高 KCl 对器官型培养物进行去极化，探讨了持续膜去极化如何影响颗粒细胞的发育和成熟过程。去极化保留了颗粒细胞的发育过程直至未成熟树突形成阶段，但阻止了颗粒细胞突触形成的成熟过程。重要的是，这种对颗粒细胞终末成熟的阻断可以通过钙调神经磷酸酶及其特异性抑制剂的失活而逆转。这项研究表明，膜电位及其下游钙调磷酸酶信号传导的改变在触发出生后发育的颗粒细胞突触组织的成熟程序中发挥着关键作用。
At the early postnatal period, cerebellar granule cells proliferate, differentiate, migrate, and finally form refined synaptic connections with mossy fibers. During this period, the resting membrane potential of immature granule cells is relatively depolarized, but it becomes hyperpolarized in mature cells. This investigation was conducted to examine the role of this alteration in membrane potential and its downstream signaling mechanism in development and maturation of granule cells. Experiments were designed to precisely characterize the ontogenic processes of developing granule cells by combining organotypic cerebellar cultures with the specific expression of EGFP (enhanced green fluorescent protein) in granule cells by use of DNA transfection. Multiple approaches using morphology, electrophysiology, and immunohistochemistry demonstrated that granule cells developed and matured at the physiological KCl concentration in organotypic cultures in a temporally regulated manner. We addressed how persistent membrane depolarization influences the developmental and maturation processes of granule cells by depolarizing organotypic cultures with high KCl. Depolarization preserved the developmental processes of granule cells up to the stage of formation of immature dendrites but prevented the maturation processes for synaptic formation by granule cells. Importantly, this blockade of the terminal maturation of granule cells was reversed by inactivation of calcineurin with its specific inhibitor. This investigation has demonstrated that alteration of the membrane potential and its downstream calcineurin signaling play a pivotal role in triggering the maturation program for the synaptic organization of postnatally developing granule cells.