Improved strategy for mass spectrometric analysis of neurotransmitters/neuromodulators from identifiable neurons of the fruit fly, Drosophila melanogaster

• Applicant: Privatdozentin Dr. Susanne Neupert, Ph.D.
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 251471599

Messenger molecules released from neurons play key roles in cell-cell communication within neuronal networks. Their structural diversity and spatial localization in the nervous system reflect their influence in an extensive variety of physiological processes. The knowledge of the qualitative and quantitative chemical composition of these signalling molecules in individual cells is crucial for studying and understanding behavioural patterns and physiological processes in organisms. Single cell analysis by mass spectrometry allows for precise determination of neuroactive substances at the cellular level. Previous studies have shown that the analyses of neuroactive substances are even possible for single cells in insects as small as Drosophila. However, these mass spectrometry based analyses are optimized for neuropeptides, which often co-localize with classical transmitters such as acetylcholine (ACh), GABA or glutamate. This project aims to fill the gap and add metabolomics measurements via capillary electrophoresis (CE) electrospray (ESI) mass spectrometry (MS) to the MS-based peptide detection; this assay suite allows the investigation of neuropeptides and biogenic amines/classical neurotransmitters from selected individual cells of the fruit fly Drosophila melanogaster. Numerous studies reveal remarkable similarities between invertebrates and vertebrates in their cellular and molecular mechanisms so that the genetic manipulable insect Drosophila melanogaster provides an ideal model for studying the composition of signaling molecules in individual cells. In this project, we will establish a robust and reproducible strategy for the characterization and quantification of the biogenic amines octopamine, tyramine and serotonin from individual isolated cells of Drosophila melanogaster. Subsequently we will apply this technique to measure biogenic amine levels in defined single cells depending on the animal physiological state. To interfere with synaptic transmission during sample preparations, we will overexpress the temperature-sensitive dynamin allele shibire (shi[ts]) using the GAL4/UAS system. Dynamin is crucial for proper vesicle scission, and the use of shits1 will allow us to reduce dissection-induced transmitter and neuromodulator release.Finally we will combine our single-cell CE-ESI MS strategy for classical neurotransmitter detection with MALDI-TOF MS-based neuropeptide identification in one workflow. Such a comprehensive signal molecule map on single cell level leads to a more detailed understanding of the complex functions of neuronal circuits. The representation of all obtained mass spectrometric and neuroanatomical data will be summarized in a 3D-Drosophila standard brain.

• DFG Programme: Research Grants