title: Functional Characterization of a Vesicular Glutamate Transporter in an Interneuron That Makes Excitatory and Inhibitory Synaptic Connections in a Molluscan Neural Circuit.
abstract: Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029 jingj01@live.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Jing
authors.affiliation: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Vera
authors.initials: V
authors.lastname: Alexeeva
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and.
authors.forename: Song-An
authors.initials: SA
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and.
authors.forename: Ke
authors.initials: K
authors.lastname: Yu
authors.affiliation: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Due
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and.
authors.forename: Li-Nuo
authors.identifier: http://orcid.org/0000-0001-7307-0980
authors.initials: LN
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University School of Life Sciences, Nanjing, Jiangsu 210093, China and.
authors.forename: Dan-Dan
authors.identifier: http://orcid.org/0000-0002-6031-2386
authors.initials: DD
authors.lastname: Liu
authors.affiliation: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Cropper
authors.affiliation: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Ferdinand S
authors.initials: FS
authors.lastname: Vilim
authors.affiliation: Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Klaudiusz R
authors.initials: KR
authors.lastname: Weiss
mesh_terms: D000818:Animals; D001048:Aplysia; D019556:COS Cells; D002522:Chlorocebus aethiops; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology*; D005260:Female; D053444:Inhibitory Postsynaptic Potentials / Q000502:physiology*; D007395:Interneurons / Q000502:physiology*; D008297:Male; D009415:Nerve Net / Q000737:chemistry / Q000502:physiology*; D010802:Phylogeny; D050496:Vesicular Glutamate Transport Proteins / Q000032:analysis / Q000502:physiology*
chemical_list: D050496:Vesicular Glutamate Transport Proteins
keywords: Aplysia; central pattern generator; feeding; glutamate; interneuron; vesicular glutamate transporter