Suriano Christos Michael
Biology Department, Wesleyan University, Middletown, CT, 06459, USA.
Princeton Neuroscience Institute, Princeton University, Princeton, NJ, 08540, USA.
Heliyon. 2019 Jan 31;5(1):e01176. doi: 10.1016/j.heliyon.2019.e01176. eCollection 2019 Jan.
The Inositol 1,4,5-trisphosphate receptor type 1 protein (Ip3r1) performs an essential role for the induction of cerebellar long-term depression. Here, I describe the use of RT-PCR, qPCR, western blotting and immunohistochemistry to assay gene expression and localize Ip3r1 protein in the hindbrain of the elasmobranch fish, . Elasmobranchs are representatives of the most basal, yet extant lineage of gnathostomes, or jawed vertebrates. The cerebellum is a synapomorphy for gnathostomes and thus elasmobranch cerebellar physiology may serve as a proxy for the ancestral state of other jawed vertebrates. is selectively expressed in the cerebellum of the little skate and the resultant protein is localized to Purkinje cells. If Ip3r1 performs the same functions in the skate cerebellum as in the mammalian cerebellum, then parallel fiber-Purkinje cell long-term depression through Ip3r1 mediated intracellular calcium regulation may be a conserved feature of cerebellar physiology. Cerebellum and surrounding hindbrain regions termed cerebellum-like structures share a common developmental genetic toolkit. expression was lowly detected in cerebellum-like structures indicating that although generatively homologous, the cerebellum and cerebellum-like structures do not share a complete overlap of common expression. Because of the little skate's important phylogenetic placement, performing molecular methodologies to assay targeted gene expression and determine protein localization in the hindbrain can be valuable for our understanding of cerebellar evolution and comparative neural development.
1,4,5-三磷酸肌醇受体1型蛋白(Ip3r1)在小脑长时程抑制的诱导过程中发挥着重要作用。在此,我描述了运用逆转录聚合酶链反应(RT-PCR)、定量聚合酶链反应(qPCR)、蛋白质免疫印迹法和免疫组织化学来检测基因表达并在板鳃亚纲鱼类的后脑定位Ip3r1蛋白。板鳃亚纲动物是有颌类脊椎动物中最原始但仍现存的谱系的代表。小脑是有颌类脊椎动物的一个共源性状,因此板鳃亚纲动物的小脑生理学可作为其他有颌类脊椎动物祖先状态的一个代表。Ip3r1在小斑鳐的小脑中选择性表达,所产生的蛋白质定位于浦肯野细胞。如果Ip3r1在鳐鱼小脑中发挥的功能与在哺乳动物小脑中相同,那么通过Ip3r1介导的细胞内钙调节实现的平行纤维 - 浦肯野细胞长时程抑制可能是小脑生理学的一个保守特征。小脑和称为类小脑结构的周围后脑区域共享一个共同的发育遗传工具包。在类小脑结构中低水平检测到Ip3r1表达,这表明尽管在发育上具有同源性,但小脑和类小脑结构在共同表达上并不完全重叠。由于小斑鳐重要的系统发育位置,运用分子方法检测后脑中有针对性的基因表达并确定蛋白质定位,对于我们理解小脑进化和比较神经发育可能具有重要价值。