Graduate Program of Neuroscience, The University of Western Ontario, London, N6A 5B7, Canada.
Robarts Research Institute, London, N6A 5B7, Canada.
Cerebellum. 2020 Aug;19(4):510-526. doi: 10.1007/s12311-020-01125-7.
Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS mice at postnatal day 7 (PD7), 2 weeks (2W), and 7 weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.
一氧化氮（NO），特别是来源于神经元型一氧化氮合酶（nNOS）的一氧化氮，是浦肯野神经元（PNs）中突触传递的一种公认的调节剂，控制着运动学习和协调等基本过程。之前的表型分析显示，尽管 nNOS 小鼠表现出明显的共济失调行为，但神经元型一氧化氮合酶缺失（nNOS）和野生型（WT）成年雄性小鼠之间的小脑结构相似。然而，一项研究尚未描述 nNOS 小鼠发育过程中 PN 的分子结构及其兴奋性输入。本研究首次使用免疫组织化学和免疫印迹法探索 nNOS 小鼠小脑内的形态异常。本研究旨在研究 WT 和 nNOS 小鼠在出生后第 7 天（PD7）、2 周（2W）和 7 周（7W）时 PN 树突形态以及代谢型谷氨酸受体 1（mGluR1）、囊泡谷氨酸转运体 1 和 2（vGluT1 和 vGluT2）、基质相互作用分子 1（STIM1）和钙蛋白酶-1 在 PN 中的表达。结果表明，nNOS 小脑在 PD7 时 PN 树突分支减少，而在成年时出现异常树突棘形成。nNOS 小脑在整个发育过程中 mGluR1 的总蛋白表达减少，而 vGluT2、STIM1 和钙蛋白酶-1 的表达显著增加。在 WT 切片中用 NOS 抑制剂 L-NAME 处理可增加钙蛋白酶-1 的表达，而用 NO 供体 NOC-18 处理 nNOS 小脑切片可降低钙蛋白酶-1 的表达。此外，mGluR1 激动剂 DHPG 可增加 WT 切片中的钙蛋白酶-1，但不能增加 nNOS 切片中的钙蛋白酶-1。综上所述，这些结果表明 nNOS/NO 信号在 PN 发育中具有新的作用，特别是通过调节 mGluR1 起始的钙信号机制。
