Neuromediators in the developing olfactory system: 3D-reconstruction towards a functional understanding
During ontogenesis of holometabolic insects like butterflies (Lepidoptera) life circumstances change radically. While larval life is dominated by eating and growing, adult animals need to cope with quite different properties of sensory stimuli, especially during flight. This asks for a complete rebu...
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|Zusammenfassung:||During ontogenesis of holometabolic insects like butterflies (Lepidoptera) life circumstances change radically. While larval life is dominated by eating and growing, adult animals need to cope with quite different properties of sensory stimuli, especially during flight. This asks for a complete rebuilding of the brain during metamorphosis to fulfill adult-specific requirements. One well-established model system to examine these develomental processes is the brain of the sphinx moth Manduca sexta. Within about three weeks of pupal development the whole brain increases about ten times in size, several larval cells die, other adult-specific neuroblasts start to proliferate while other neuronal cells are restructured to deal with adult-specific tasks. Many of the underlying ontogenetic processes of outgrowing and differentiation of neural cells seem to be conserved between insects and vertebrates. On the molecular level as well as on the level of neuropilar organization exist parallels between both, which usually are more accessible in the insect model. One example would be target selection of sensory receptor neurons, which is steered by similar contact receptors. Another example is the analogous organization of olfactory neuropils into spherical glomeruli which exhibit odor-dependent activation. The gaseous signal molecule nitric oxide (NO) and its influence on neurogenesis represents another example of a conserved concept during individual development of various systems, including antennal lobe development in Manduca. The modes of action of NO are manyfold: it may influence proteins by ADP-ribosylation or S-nitrosylation, or may stimulate further intracellular signalling cascades. Most prominent example in Manduca would be the transient cGMP synthesis by NO-dependent soluble guanylyl cyclase. This cGMP in turn exhibits its own broad spectrum of downstream activation, reaching from directly dependent ion channels (cyclic nucleotide gated channels, CNGs) to protein kinases and phosphodiesterases. Within the framework of our working hypothesis the release of neuropeptides in developing local interneurons is mediated by selective cGMP production during a defined time window. This allows for specific refinement of synaptic contacts coming from a previous unspecific developmental signal. Our existing neuropeptide data was extended by anatomical and pharmacological examinations regarding allatotropin and further characterization of the lateral cell group by MALDI-TOF mass spectrometry. The emphasis of this dissertation however was laid on further characterization of the NO/cGMP signalling pathway during antennal lobe develoment of Manduca sexta. First, the cell group in question was examined towards its ability to extend the NO signal by Cu,Zn superoxide dismutase expression, followed by a test for cell death during antennal lobe development. Since no dying cells were found throughout antennal lobe development, a stable cell population could be assumed. Now, the NO/cGMP signalling pathway was further examined with respect to temporal occurrence, specificity, and number of cells involved; additionally, its possible mode of action on neuronal development was pointed out by integrating previous work. Within this framework, 3D-reconstruction based volumetry was used, which proved to be a powerful tool to answer other questions during antennal lobe development as well. Prerequisite herefore was, however, the establishment of a suitable protocol, together with the definition of easily and reproducably identifyable neuropilar regions in the antennal lobe during development. After introduction of this protocol, an extention onto whole brain neuropils was obvious, which was done in a supervised diploma work. In this context, a Manduca standard brain was created and used to volumetrically examine brain sex dimorphism. In a cooperation project, anatomical expertise on the antennal lobe system was applied by identification and classification of intracellulary recorded projection neurons, combined wtih a 4D representation of the data obtained.|