Gap junction regulation controls firing synchrony in neurons that initiate reproduction

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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.