Single cell analysis within neuronal networks

My research group “Single cell analysis within neuronal networks” belongs to the graduate school “clocks” at the University Kassel. We are working on a broad array of projects that span analytical chemistry, entomology to neuroscience and neuroethology focusing on two classes of messenger molecules: neuropeptides and biogenic amines. Both neuroactive substance classes are released from neurons and play key roles in cell-cell communication in neuronal networks. Their occurrence, structural diversity and spatial localization in the nervous system reflect their influence in an extensive variety of physiological processes, neural dysfunctions and behavioral patterns. To study principal questions about their function in complex neuronal circuits, appropriate methods and workflows for precise determination and quantification of these neuroactive substances in tissues, organs, cell population up to the cellular and subcellular level have been available and novel toolsets are constantly developed and optimized. Our group research contains three lines of research:

1. Neuropeptidomics up to single cell level

Neuropeptidomics indicates the measurement and identification of the neuropeptide composition from a single cell, defined tissue samples and extracts from the brain or neuronal tissue. Obviously, peptide characterization has a much longer history, but the ability to generate lists of hundreds of neuropeptides and peptide precursor products from a tissue or a single cell sample characterizes the new generation of peptide identification. In our team, we are using next-generation omics-methods mainly based on high-resolution mass spectrometry and ultra-performance liquid chromatography coupled with electrospray ionization to enable the detection and quantification of neuropeptides in ultra-high sensitivity from tissue extracts up to single cell level as well as on transcriptional level using single cell transcriptomics to examines the gene expression level particular GPCR- and neuropeptide coding genes of individual cells. The advanced neuroanalytical tools allow me to (A) study the peptidergic equipment of an organism as completely as possible; (B) analyze differential expression events of the genome influence (“normal”) cell molecular processes; (C) uncover differential peptide processing events within a neuronal tissue or cell population; (D) map neuroactive substances on single cell level (E) cell heterogeneity as well as (F) investigate co-localizations of signaling molecules encoded by different genes. Specific questions address what molecules are present in specific cells, cell populations, tissues, organs and networks, how they change based on behavior or exposure to drugs, as well as their function.

2. Physiology of feeding and sleep in Drosophila melanogaster on circadian timing and on responses to environmental stress on single cell level.

The diurnal adult fruitfly Drosophila melanogaster expresses circadian and ultradian rhythms of rest (sleep) and activity (locomotion and feeding) with activity peaks at dusk and dawn. Neuropeptides and biogenic amines play key roles in the timing of cell-cell communication in neuronal networks that regulate e.g. feeding and sleeping. A certain balance of circadian and ultradian rhythms of rest (sleep) and activity coordinated by neuropeptides and biogenic amines is important in refining brain circuitry. Instead of mostly relaying on genetic techniques, we approach this research questions with a different biochemical tool set. To gain new insights into the roles of neuropeptides and biogenic amines in the control of feeding and sleep behavior, we focus our research on the bioanalytical analysis of individual neurons. This includes identification of release sites using different MS -based tools to uncover the set of processed and/or stored signaling molecules, including sequence and structural analysis, posttranslational peptide modifications, co-localizations and differential processing events within single neurons. Besides of the analysis of qualitative signaling, our research concentrates also on quantitative evaluation of these molecules on the single cell level. Our new neurochemical insights gained on single cell studies in flies and its regulation via endogenous rhythms and environmental stress will support the understanding cellular and molecular mechanisms of rest and activity also in vertebrates and thus to help developing of pharmacological treatments.

3. Single-cell analysis: development and optimization of mass spectrometry-based tools for neuroscience research

A neuron can contain both neuropeptides and classical neurotransmitter (e.g. biogenic amine). Neuropeptide profiling using MALDI-TOF MS from single cells is meanwhile a popular used tool in neurobiology, however, the analysis of biogenic amines from individual cells using MS-based tools, particularly with MALDI-TOF MS, is only the beginning. Biogenic amines are deamination products of amino acids and have been associated with many physiological and behavioral processes. In order to understand functional roles of amines in neuronal circuits in more detail, the need for method development and optimization is obvious in order to obtain new neurochemical insights in neuronal circuits, physiological processes and behavioral pattern of organisms. The continually expanding requirements in neuroscience research to measure volume-limited samples such as single cell or subcellular domains as completely as possible require the continuous development of novel approaches with a higher-throughput and greater sensitivity. Therefore, I focused my research on the developing and optimizing MS-based strategies, mainly focused on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS and sample preparation protocols which allow the identification, quantification and the analysis of evaluation of co-existing events of neuropeptide and biogenic amines from single insect neurons, including their release sites. Particularly, measurements of neuronal release sites within neuronal circuits are still lacking. With imaging mass spectrometry, a tool to investigate the spatial distribution of molecules of interests throughout the tissue section, we have the great advantage to study the distribution of neuroactive substances not only on the soma but also in the axon and release site.

While the technology and approaches are developed using insect model systems as a test-bed, the technology advancement resulting from our research is widely applicable to the large-scale analysis of peptides and biogenic amines in many biological systems, including those of mammalian and humans to understand cellular and molecular processes in embryogenesis, regeneration and the organization of neuronal networks. Understanding the role of single cells in the etiology and development of disease can be essential for the development of pharmacological treatments for a variety of illnesses, including cardiovascular diseases, diabetes, mental illness, stroke and cancer.

PD Dr. Susanne Neupert

full member

Neupert, Susanne
+49 561 804-4781
+49 561 804-4146
Universität Kassel
FB 10 Naturwissenschaften
Abteilung Tierphysiologie
Heinrich-Plett-Straße 40
34132 Kassel