Members of the Phylum Cnidaria (sea anemones, corals, jellyfish) represent one of the earliest groups of animals to possess a nervous system. As such, they provide a baseline from which to track the evolution of nervous systems as a whole, and of the molecular building blocks of the nervous system. Despite considerable knowledge about the capabilities of the nervous systems of these animals, very little is known about one of the most fundamental aspects of the neurobiology of these animals, specifically the identity of the neurotransmitters used by these animals at the fast chemical synapses that are so prevalent in their nervous systems. To address this question the researchers will employ a combination of electrophysiological and cell and molecular biological techniques to identify the neurotransmitter at synapses within the nervous system of the jellyfish Cyanea capillata. This project will contribute enormously to understanding of nervous system evolution and will provide important information about the evolutionary lineages of the neurotransmitters used by higher nervous systems, including that of humans. The broader impacts of this project promise to be considerable inasmuch as undergraduates participating in the Whitney Laboratory's REU program will be included in the work. Furthermore, the Whitney Laboratory has an extensive program of educational outreach to diverse sectors of the local community, including grade schools. The results from this project will be conveyed to these audiences by displays and other forms of communication, thereby contributing to their training and their appreciation and understanding of science.
Members of the Phylum Cnidaria, the sea anemones, corals and jellyfish occupy an important position in the evolution of the nervous system because they are one of the earliest groups of animals to possess a nervous system. Importantly, they provide useful preparations with which to study the cellular properties of the "early nervous system" and in that sense are likely to provide far more information than members of the Phylum Ctenophora, the comb jellies, which are currently thought to be the earliest of all animals to possess a nervous system. This study was designed to resolve a long-standing question about the earliest nervous system; what kind of chemical(s) (neurotransmitter(s)) were used by the earliest nervous systems for communication at fast chemical synapses. The bidirectional chemical synapses between neurons of the Motor Nerve Net (MNN) of the jellyfish Cyanea capillata provide an unprecedented opportunity for such a study inasmuch as they allow one to record from both the pre- and post-synaptic neurons. Preliminary data had suggested that the amino acid, taurine, or a close analog, was the neurotransmitter at these synapses since applications of taurine to exposed synapses evoked large depolarizations that were functionally the same as the normal synaptic event. The goals of this project were to confirm this preliminary finding and, specifically, to determine if taurine is present in the neurons, that it is released from the synapses in an activity- dependent manner, that appropriate receptors are present on the neurons to detect that taurine and that mechanisms to terminate the action of the neurotransmitter (e.g. uptake mechanisms, enzymes or transporters) are present at the synapses. We employed a variety of modern cell and molecular biological techniques in this study. Using capillary electrophoresis (CE) we confirmed that taurine is present in MNN neurons along with measurable amounts of GABA, phenylalanine, serine and glycine. However, we were unable to confirm that taurine is released from the neurons in an activity-dependent manner. In our search for mechanisms that might serve to terminate the action of released taurine we cloned a putative neurotransmitter transporter from MNN mRNA. This molecule, a member of the SLC6 family of transporters was mapped to the MNN neurons by both in situ hybridization and immunocytochemistry, but were not able to demonstrate that this molecule actually transports taurine. To facilitate the search for neurotransmitter receptors in MNN neurons we used Next Generation sequencing of mRNA. A challenge in working with this species is that its genome has not yet been completed. In the absence of a reference genome, we developed a new method for reconstructing and assembling gene fragments (the product of the Next Generation sequencing), functionally annotating them and estimating their abundance. Unfortunately, no neurotransmitter receptor candidates were revealed by this analysis. With respect to Broader Impacts, this project provided training to two REU students, Ms. Kassandra Ferguson and Ms. Cassandra Newkirk, both of whom are currently enrolled in Ph.D. programs at Florida State University and the University of Florida, respectively.
