Organisms need to constantly adapt their behavior to the changing environment as well as react towards changes in their internal state. The nervous system perceives and processes such stimuli and coordinates the corresponding reactions of the body. This system is based on regulated cell-cell communication, utilizing a wide range of different chemical signaling molecules and receptors. If one wants to fully grasp how neural circuits process, modulate and relay incoming information, then the involved neuroactive substances, their cellular distribution, temporal and
quantitative dynamics have to be analyzed on single cell resolution. Single cell mass spectrometry (SCMS) allows the interrogation of chemical profiles from individual cells,
including neuroactive substances such as neuropeptides and biogenic amines. Matrix assisted laser desorption/ionization – time-of-flight mass spectrometry (MALDI-TOF MS) has established itself as a fast and reliable tool for the analysis of neuropeptides from single neurons of invertebrates and vertebrates alike. However, the detection of small signaling molecules, such as biogenic monoamines, by MALDI-TOF SCMS has been challenging. Biogenic monoamines play key roles in orchestrating and modulating neural circuits, therefore a MALDI-TOF SCMS based method for their detection and quantification is highly desirable.
Additionally, biogenic monoamines can be co-localized with neuropeptides. Therefore the development of a MALDI-TOF SCMS based method capable of detecting both neuroactive substances would help to reveal such overlapping expression profiles. In the current thesis, I focused on the development of a MALDI-TOF SCMS based method that
allows the detection and quantification of biogenic monoamines from single somata of insect neurons. The study focused on the insect octopaminergic/tyraminergic system, with an emphasis on octopamine (OA), which is considered to be homologous to the vertebrate noradrenalin/adrenalin system. By using chemical derivatization of amine moieties of OA and tyramine (TA) and an optimized sample preparation, I was able to lower the respective detection limits to single cell concentrations. Additionally, I could show that the chemical derivatization does not interfere with the detection of neuropeptides from the same sample, hence allowing the simultaneous detection of both substance classes. Further, I could show that absolute quantification of OA and TA is possible from single cell sample volumes using isotopically labeled synthetic standards. I used the developed protocol for the qualitative and quantitative
analysis of OA/TA from genetically labeled and manually microdissected somata of interneurons from the fruit fly Drosophila melanogaster. Using the newly developed approach,
I analyzed intracellular OA/TA ratios, compared somatic OA titers between sexes and two different OAergic cell clusters and revealed that prolonged cooling of animals has an increasing effect on detectable OA titers in the analyzed neurons.
Furthermore, I used the developed protocol to analyze changes in somatic OA titers of aggression modulating OAergic neurons from the gnathal ganglion in socially naive and experienced adult male D. melanogaster. I could show that the somatic OA titer increases in these neurons when flies had social contact with the same sex compared to naive flies, which is possibly mediated by an input from pheromone detecting gustatory receptor neurons. To my knowledge, this is the first study to report a quantified increase of a somatic biogenic monoamine titer detected directly from individual isolated neurons of intact insect brains
between two behavioral states by mass spectrometric analysis.
In a collaborative study, I employed the developed protocol to intracellular recorded descending dorsal unpaired median neurons from the Indian stick insect Carausius morosus and was able to confirm that these neurons contain OA and TA and thus could be OAergic. Finally, as a starting point in an effort to create a map of neuropeptidergic neurons and their
repertoire of neuroactive substances in adult D. melanogaster, I was involved in the analysis of single genetically labeled neuropeptidergic neuron somata using MALDI-TOF SCMS. In summary, we could describe a total of 10 different cell types characterized by their expressed
neuropeptides and their location in the CNS. Future studies will focus on analyzing these cell types towards potential co-localized aminergic transmitters using the developed protocol
