Gap junction regulation controls firing synchrony in neurons that initiate reproduction

我的工作涉及神经元如何协调它们的电活动来启动行为。我研究控制加州海蜗牛繁殖的袋状细胞神经元。在短暂的刺激后，这组神经元经历了30分钟的爆发或后放电，并向血液中分泌激素来触发产卵行为。******在后放电过程中，袋细胞神经元通过同步发射快速电压峰（称为动作电位）释放激素。这种活动的同步是通过间隙连接实现的，间隙连接是由一种叫做连接蛋白（脊椎动物）或连接蛋白（无脊椎动物）的蛋白质构成的细胞间通道。间隙连接通过允许电流在细胞之间流动来电连接神经元。******在我目前的NSERC资助下（RGPIN 386664-2010），我们鉴定了海兔的肠连蛋白基因，并制造了三种在袋细胞神经元中发现的肠连蛋白的抗体。我们还使用电生理学（电监测）来记录通过间隙连接的电流，发现偶联被称为激酶的酶抑制，这些酶在放电后被激活，特别是蛋白激酶C和钙调蛋白激酶。激酶通过磷酸化改变蛋白质，即将磷酸盐附着在特定的氨基酸上。抑制间隙连接使袋状细胞神经元更少“渗漏”，对兴奋更敏感。******我建议对改变间隙连接如何对同步神经元活动和触发生殖至关重要进行多层次调查：******在分子水平上，我们将使用抗体和磷酸盐染色来检测哪些内联蛋白在后放电过程中被磷酸化。我们还将突变（基因移除）可能在肠内联蛋白上磷酸化的氨基酸，然后在缺乏间隙连接的细胞中表达（基因插入）这些肠内联蛋白，并使用电生理学来观察激酶的抑制是否丢失。******在神经元网络水平上，我们将使用电生理学和计算机建模来检查动作电位在激酶抑制偶联之前和之后如何通过成对和组袋细胞神经元移动。******在动物层面，在繁殖季节，后发放电更容易唤起，电突触更强。我们将使用聚合酶链反应（一种基因测定）和我们的抗体来确定innexins是否随季节变化。动物也将受到化学压力，这阻碍了繁殖，以测试对偶联和内联蛋白磷酸化的影响。******缝隙连接存在于所有动物中；因此，我的研究将通过推进对脑功能电耦合的理解来影响神经科学领域。随着知识的增强，我的计划将通过培养研究生和本科生在收集、分析和传播用多种现代生物技术收集的数据方面对加拿大有益。

My work concerns how neurons coordinate their electrical activity to initiate behaviour. I study the bag cells neurons, which control reproduction in the sea snail, Aplysia californica. After brief stimulation, this group of neurons undergoes a 30-min burst, or afterdischarge, and secretes hormone into the blood to trigger egg-laying behaviour.******During the afterdischarge, bag cell neurons release hormone by synchronously firing rapid spikes in voltage, known as action potentials, across their membrane. This synchrony of activity is achieved via gap junctions, which are cell-to-cell channels made from proteins called connexins (for vertebrates) or innexins (for invertebrates). Gap junctions electrically couple neurons by permitting current flow between cells.******Under my current NSERC grant (RGPIN 386664-2010), we identified Aplysia innexin genes and made antibodies to three innexin proteins found in bag cell neurons. We also used electrophysiology (electrical monitoring) to record current passing through gap junctions, and found that coupling was suppressed by enzymes, called kinases, activated during the afterdischarge, specifically, protein kinase C and calmodulin kinase. Kinases alter proteins by phosphorylation, ie, attaching phosphates to specific amino acids. Suppressing gap junctions renders bag cell neurons less "leaky" and more responsive to excitation.******I propose a multi-level investigation of how altering gap junctions is crucial to synchronizing neuronal activity and triggering reproduction:******At the molecular level, we will use our antibodies and a phosphate stain to detect which innexins are phosphorylated during the afterdischarge. We will also mutate (genetically remove) amino acids that are potentially phosphorylated on the innexins, then express (genetically insert) those innexins in cells which lack gap junctions, and use electrophysiology to see if suppression by kinases is lost.******At the neuronal network level, we will use electrophysiology and computer modelling to examine how action potentials move through pairs and groups of bag cell neurons before and after suppression of coupling by kinases.******At the animal level, the afterdischarge is easier to evoke and electrical synapses are stronger during the breeding season. We will use polymerase chain reaction (a genetic assay) and our antibodies to ascertain if innexins change with season. Animals will also be chemically stressed, which impedes reproduction, to test the effect on coupling and innexin phosphorylation.******Gap junctions are found in all animals; thus, my study will impact the field of neuroscience by advancing the understanding of electrical coupling in brain function. Along with this enhancement of knowledge, my plan will benefit Canada by training graduate and undergraduate HQP in the collection, analysis, and dissemination of data gathered with a diversity of modern biological techniques.