| In an attempt to probe insect neurotransmission at a molecular level, neurotransmitter transporters from the mosquito Aedes aegypti and tobacco hornworm Manduca sexta were isolated and characterized with regards to localization, function, pharmacology, and electrophysiology. Three classes of neurotransmitter transporters were studied: (1) a M. sexta γ-aminobutyric acid (GABA) transporter (MasGAT) belonging to the Na+/Cl− dependent plasma membrane transporter family, (2) an Ae. aegypti excitatory amino acid (EAAT) transporter (AeaEAAT), falling under the Na+/K + dependent plasma membrane transporter family, and (3) a M. sexta vesicular acetylcholine transporter homologue, a part of the vesicular monoamine family of transporters.; Immunocytochemical localization of MasGAT using a specific antibody to a variable region of the protein, demonstrated abundance in the neuropile regions of the brain and ganglia throughout the course of development. Immunoreactivity was evident from as early as the second-day embryo, and sustained through adulthood. It strongly mirrored the staining pattern for GABA in M. sexta as well as for the GABA receptor subunits in D. melanogaster that had been previously observed.; A thorough functional characterization of AeaEAAT, having 40–50% identity to other known EAATs from both vertebrates and invertebrates, demonstrated it as the functional homologue of human EAAT3. It transported L-glutamate, L- and D-aspartate with high affinity, and exhibited current responses to application of L-cysteine, L-glutamine, and L-asparagine. Like its mammalian homologues, AeaEAAT of mediated a substrate-elicited anion conductance that was not required for functional substrate transport. Studies into the localization of this protein illustrated highest levels in the adult thorax, particularly in the thoracic ganglia.; Finally, an antibody was developed to a variable region of the cloned MasVTR to localize cholinergic cells in M. sexta. MasVTR had 40–50% amino acid identity to characterized vertebrate acetylcholine transporters, with ca. 70% identity to a D. melanogaster homologue. Immunolocalization of the larval 5th instar ventral nerve cord showed staining of discreet cell clusters in the brain and thoracic ganglia.; Collectively, this thesis provides the nature of the chemical architecture of the insect nervous system from the standpoint of proteins involved in transporting the neurotransmitters. |
