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以启动行为。 我研究的是袋状细胞神经元，它控制着加利福尼亚海蜗牛的繁殖。 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，并将激素分泌到血液中以触发产卵行为。在后放电过程中，囊细胞神经元通过同步触发跨膜的快速电压尖峰（称为动作电位）来释放激素。 这种活动的同步是通过间隙连接实现的，间隙连接是由称为连接蛋白（脊椎动物）或连接蛋白（无脊椎动物）的蛋白质制成的细胞与细胞通道。 缝隙连接通过允许电流在细胞之间流动来电耦合神经元。在我目前的NSERC资助（RGPIN 386664-2010）下，我们鉴定了Aussia innexin基因，并制备了袋细胞神经元中发现的三种innexin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，并发现偶联被称为激酶的酶抑制，这些酶在后放电期间被激活，特别是蛋白激酶C和钙调蛋白激酶。 激酶通过磷酸化作用改变蛋白质，即将磷酸连接到特定的氨基酸上。 抑制缝隙连接使袋细胞神经元更少“泄漏”，对兴奋更敏感。我提出了一个多层次的调查如何改变缝隙连接是至关重要的同步神经元活动和触发生殖：** 在分子水平上，我们将使用我们的抗体和磷酸盐染色检测哪些innexins磷酸化在后放电。 我们还将突变（遗传去除）可能在连接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（遗传插入）这些连接蛋白，并使用电生理学来观察激酶的抑制是否消失。在神经元网络水平，我们将使用电生理学和计算机建模来研究在激酶抑制偶联之前和之后，动作电位如何通过成对和成组的袋细胞神经元移动。在动物水平上，在繁殖季节，后放电更容易引起，电突触更强。 我们将使用聚合酶链反应（一种基因检测）和我们的抗体来确定连接蛋白是否随季节而变化。 动物也将受到化学应激，这会阻碍繁殖，以测试对偶联和连接蛋白磷酸化的影响。缝隙连接存在于所有动物中;因此，我的研究将通过促进对大脑功能中电耦合的理解来影响神经科学领域。 沿着知识的提高，我的计划将通过培训研究生和本科生HQP收集，分析和传播用各种现代生物技术收集的数据，使加拿大受益。

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.