@phdthesis{HornneeBunz2020,
  author    = {Horn [n{\´e}e Bunz], Melanie},
  title     = {The impact of Drosophila melanogaster`s endogenous clock on fitness: Influence of day length, humidity and food composition},
  doi       = {10.25972/OPUS-21141},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211415},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {We are living in a system that underlies permanent environmental changes due to the rotation of our planet. These changes are rhythmic with the most prominent one having a period of about 24 hours, but also shorter and longer rhythms characterize our environment. To cope with the ever-changing environmental conditions, it is thought to be beneficial if an organism can track and anticipate these changes. The so called endogenous clocks enable this and might provide a fitness advantage. To investigate and unravel the mechanism of endogenous clocks Chronobiologists have used different model organisms. In this thesis Drosophila melanogaster was used as model organism with its about 150 clock neurons representing the main endogenous clock of the fly in the central brain. The molecular mechanisms and the interlocked feedback loops with the main circadian key players like period, timeless, clock or cycle are under investigation since the 1970s and are characterized quite well so far. But the impact of a functional endogenous clock in combination with diverse factors and the resulting fitness advantages were analysed in only a few studies and remains for the most part unknown. Therefore the aim of this thesis was to unravel the impact of Drosophila melanogaster`s endogenous clock on the fitness of the fly. To achieve this goal different factors - like day length, humidity and food composition - were analyzed in wild type CS and three different period mutants, namely perL, perS and per01, that carry a point mutation altering or abolishing the free-running period of the fruit fly as well as a second arrhythmic strain, clkAR. In competition assay experiments wild type and clock mutant flies competed for up to 63 generations under a normal 24 hour rhythm with 12 hours light/day and 12 hours darkness/night (LD12:12) or T-cycles with 19 or 29 hours, according to the mutants free-running period, or constant light (LL) in case of the arrhythmic mutant as well as under natural-like outdoor conditions in two consecutive years. Overall the wild type CS strain was outcompeting the clock mutant strains independent of the environmental conditions. As the perL fly strain elongated their free-running period, the competition experiments were repeated with naturally cantonized new fly strains. With these experiments it could be shown that the genetic background of the fly strains - which are kept for decades in the lab, with backcrosses every few years - is very important and influences the fitness of flies. But also the day length impacts the fitness of the flies, enabling them to persist in higher percentage in a population under competition. Further factors that might influence the survival in a competing population were investigated, like e.g. mating preferences and locomotor activity of homo- and heterozygous females or sperm number of males transferred per mating. But these factors can still not explain the results in total and play no or only minor roles and show the complexity of the whole system with still unknown characteristics. Furthermore populations of flies were recorded to see if the flies exhibit a common locomotor activity pattern or not and indeed a population activity pattern could be recorded for the first time and social contact as a Zeitgeber could be verified for Drosophila melanogaster. In addition humidity and its impact on the flies´ fitness as well as a potential Zeitgeber was examined in this thesis. The flies experienced different relative humidities for eclosion and wing expansion and humidity cycle phase shifting experiments were performed to address these two different questions of fitness impact and potential Zeitgeber. The fruit fly usually ecloses in the morning hours when the relative humidity is quite high and the general assumption was that they do so to prevent desiccation. The results of this thesis were quite clear and demonstrate that the relative humidity has no great effect on the fitness of the flies according to successful eclosion or wing expansion and that temperature might be the more important factor. In the humidity cycle phase shifting experiments it could be revealed that relative humidity cannot act as a Zeitgeber for Drosophila melanogaster, but it influences and therefore masks the activity of flies by allowing or surpressing activity at specific relative humidity values. As final experiments the lifespan of wild type and clock mutant flies was investigated under different day length and with different food qualities to unravel the impact of these factors on the fitness and therefore survival of the flies on the long run. As expected the flies with nutrient-poor minimum medium died earlier than on the nutrient-rich maximum medium, but a small effect of day length could also be seen with flies living slightly longer when they experience environmental day length conditions resembling their free-running period. The experiments also showed a fitness advantage of the wild type fly strain against the clock mutant strains for long term, but not short term (about the first 2-3 weeks). As a conclusion it can be said that genetic variation is important to be able to adapt to changing environmental conditions and to optimize fitness and therefore survival. Having a functional endogenous clock with a free-running period of about 24 hours provides fitness advantages for the fruit fly, at least under competition. The whole system is very complex and many factors - known and unknown ones - play a role in this system by interacting on different levels, e.g. physiology, metabolism and/or behavior.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Chen2018,
  author    = {Chen, Jiangtian},
  title     = {Functions of allatostatin A (AstA) and myoinhibitory peptides (MIPs) in the regulation of food intake and sleep in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156838},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{W{\"U}} cells by using newly generated \textit{Mip\textsuperscript{W{\"U}}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{W{\"U}} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}\$>\$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par},
  subject      = {AstA},
  language  = {en}
}
@phdthesis{Scholz2017,
  author    = {Scholz, Nicole},
  title     = {Genetic analyses of sensory and motoneuron physiology in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {During my PhD I studied two principal biological aspects employing Drosophila melanogaster. Therefore, this study is divided into Part I and II. Part I: Bruchpilot and Complexin interact to regulate synaptic vesicle tethering to the active zone cytomatrix At the presynaptic active zone (AZ) synaptic vesicles (SVs) are often physically linked to an electron-dense cytomatrix - a process referred to as "SV tethering". This process serves to concentrate SVs in close proximity to their release sites before contacting the SNARE complex for subsequent fusion (Hallermann and Silver, 2013). In Drosophila, the AZ protein Bruchpilot (BRP) is part of the proteinous cytomatrix at which SVs accumulate (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Intriguingly, truncation of only 1\% of the C-terminal region of BRP results in a severe defect in SV tethering to this AZ scaffold (hence named brpnude; Hallermann et al., 2010b). Consistent with these findings, cell-specific overexpression of a C-terminal BRP fragment, named mBRPC-tip (corresponds to 1\% absent in brpnude; m = mobile) phenocopied the brpnude mutant in behavioral and functional experiments. These data indicate that mBRPC-tip suffices to saturate putative SV binding sites, which induced a functional tethering deficit at motoneuronal AZs. However, the molecular identity of the BRP complement to tether SVs to the presynaptic AZ scaffold remains unknown. Moreover, within larval motoneurons membrane-attached C-terminal portions of BRP were sufficient to tether SVs to sites outside of the AZ. Based on this finding a genetic screen was designed to identify BRP interactors in vivo. This screen identified Complexin (CPX), which is known to inhibit spontaneous SV fusion and to enhance stimulus evoked SV release (Huntwork and Littleton, 2007; Cho et al., 2010; Martin et al., 2011). However, so far CPX has not been associated with a function upstream of priming/docking and release of SVs. This work provides morphological and functional evidence, which suggests that CPX promotes recruitment of SVs to the AZ and thereby curtails synaptic short-term depression. Together, the presented findings indicate a functional interaction between BRP and CPX at Drosophila AZs. Part II: The Adhesion-GPCR Latrophilin/CIRL shapes mechanosensation The calcium independent receptor of α-latrotoxin (CIRL), also named Latrophilin, represents a prototypic Adhesion class G-protein coupled-receptor (aGPCR). Initially, Latrophilin was identified based on its capacity to bind the α-component of latrotoxin (α-LTX; Davletov et al., 1996; Krasnoperov et al., 1996), which triggers massive exocytotic activity from neurons of the peripheral nervous system (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). As a result Latrophilin is considered to play a role in synaptic transmission. Later on, Latrophilins have been associated with other biological processes including tissue polarity (Langenhan et al., 2009), fertility (Pr{\"o}mel et al., 2012) and synaptogenesis (Silva et al., 2011). However, thus far its subcellular localization and the identity of endogenous ligands, two aspects crucial for the comprehension of Latrophilin's in vivo function, remain enigmatic. Drosophila contains only one latrophilin homolog, named dCirl, whose function has not been investigated thus far. This study demonstrates abundant dCirl expression throughout the nervous system of Drosophila larvae. dCirlKO animals are viable and display no defects in development and neuronal differentiation. However, dCirl appears to influence the dimension of the postsynaptic sub-synaptic reticulum (SSR), which was accompanied by an increase in the postsynaptic Discs-large abundance (DLG). In contrast, morphological and functional properties of presynaptic motoneurons were not compromised by the removal of dCirl. Instead, dCirl is required for the perception of mechanical challenges (acoustic-, tactile- and proprioceptive stimuli) through specialized mechanosensory devices, chordotonal organs (Eberl, 1999). The data indicate that dCirl modulates the sensitivity of chordotonal neurons towards mechanical stimulation and thereby adjusts their input-output relation. Genetic interaction analyses suggest that adaption of the molecular mechanotransduction machinery by dCirl may underlie this process. Together, these results uncover an unexpected function of Latrophilin/dCIRL in mechanosensation and imply general modulatory roles of aGPCR in mechanoception.},
  subject      = {Drosophila},
  language  = {en}
}
@phdthesis{Koenig2016,
  author    = {K{\"o}nig, Sebastian},
  title     = {Spatially selective visual attention in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134452},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Finding the right behavior at the right time is one of the major tasks of brains. In a natural scenery there is often an abundance of stimuli present and the brain has to separate the relevant from the irrelevant ones. Selective visual attention (SVA) is a property of higher visual systems that achieves this separation, as it allows to '[…] focus on one source of sensory input to the exclusion of others' (Luck and Mangun, 1996). There are probably several forms of SVA depending upon the criteria used for the separation, such as salience, color, location in space, novelty, or motion. Many studies have investigated SVA in humans and non-human primates. However, complex functions like attention were initially not expected to be already implemented in the brains of simple organisms like Drosophila. After a first demonstration of selective attention in the fly (Wolf and Heisenberg, 1980), it took some time until other studies included attentional mechanisms in their argumentation to explain certain behaviors of Drosophila. However, their definition and characterization of attention differed and often was ambiguous. Here, one particular form, spatially selective visual attention in the fly Drosophila is investigated. It has been shown earlier that the fly spontaneously may restrict its behavioral responses in stationary flight to the visual stimuli on one side of the visual field. On the basis of experiments of Sareen et al., (2011) it has been conjectured that the fly has a focus of attention (FoA) and that the fly responds to the visual stimuli within this area of the visual field. Whether the FoA is the adequate concept for this spatial property of SVA in the fly needs to be further discussed and is a subject also of the present study. At this stage, the concept will be used in the description of the new results expanding the characterization of SVA. This study continued the investigation of SVA during tethered flight with variable but controlled visual input and an automated primary data evaluation. This standardized paradigm allowed for analysis of wild-type behavior as well as for a comparison of several mutant and pharmacologically manipulated strains to the wild-type. Some properties of human SVA like the occurrence of externally as well as internally caused shifts of attention were found in Drosophila and it could be shown, that SVA in the fly can be externally guided and has an attention span. Additionally, a neurotransmitter and proteins, which play a significant role in SVA were discovered. Based on this, the genetic tools available for Drosophila provided the means to a first examination of cells and circuits involved in SVA. Finally, the free walk behavior of flies that had been shown to have compromised SVA was characterized. The results suggested that the observed phenotypes of SVA were not behavior specific. Covert shifts of the FoA were investigated. The FoA can be externally guided by visual cues to one or the other side of the visual field and even after the cue has disappeared it remains there for <4s. An intriguing finding of this study is the fact, that the quality of the cue determines whether it is attractive or repellent. For example a cue can be changed from being repellent (negative) to being attractive (positive) by changing its oscillation amplitude from 4° to 2°. Testing the effectiveness of cues in the upper and lower visual field separately, revealed that the perception of a cue by the fly is not exclusively based on a sum of its specifications. Because positive cueing did not have an after-effect in each of the two half-fields alone, but did so if the cue was shown in both, the fly seems to evaluate the cue for each combination of parameters specifically. Whether this evaluation of the cue changed on a trial-to-trial basis or if the cue in some cases failed to shift the FoA can at this point not be determined. Looking at the responses of the fly to the displacement of a black vertical stripe showed that they can be categorized as no responses, syn-directional responses (following the direction of motion of the stripe) and anti-directional responses (in the opposite direction of the motion of the stripe). The yaw-torque patterns of the latter bared similarities with spontaneous body saccades and they most likely represented escape attempts of the fly. Syn-directional responses, however, were genuine object responses, distinguishable by a longer latency until they were elicited and a larger amplitude. These properties as well as the distribution of response polarities were not influenced by the presence or absence of a cue. When two stripes were displaced simultaneously in opposite directions the rate of no responses increased in comparison to the displacement of a single stripe. If one of the stripes was cued, both, the responses towards and away from the side of cue resembled the syn-directional responses. Significant progress was made with the elucidation of the neuronal underpinnings of SVA. Ablation of the mushroom bodies (MB) demonstrated their requirement for SVA. Furthermore, it was shown that dopamine signaling has to be balanced between too much and too little. Either inhibiting the synthesis of dopamine or its re-uptake at the synapse via the dDAT impaired the flies' susceptibility to cueing. Using the Gal4/UAS system, cell specific expression or knockdown of the dDAT was used to scrutinize the role of MB sub-compartments in SVA. The αβ-lobes turned out to be necessary and sufficient to maintain SVA. The Gal4-line c708a labels only a subset of Kenyon cells (KC) within the αβ-lobes, αβposterior. These cells stand out, because of (A) the mesh-like arrangement of their fibers within the lobes and (B) the fact that unlike the other KCs they bypass the calyx and thereby the main source of olfactory input to the MBs, forming connections only in the posterior accessory calyx (Tanaka et al., 2008). This structure receives no or only marginal olfactory input, suggesting for it a role in tasks other than olfaction. This study shows their requirement in a visual task by demonstrating that they are necessary to uphold SVA. Restoring dDAT function in these approximately only 90 cells was probably insufficient to lower the dopamine concentration at the relevant synapses and hence a rescue failed. Alternatively, the processes mediating SVA at the αβ-lobes might require an interplay between all of their KCs. In conclusion, the results provide an initial point for future research to fully understand the localization of and circuitry required for SVA in the brain. In the experiments described so far, attention has been externally guided. However, flies are also able to internally shift their FoA without any cues from the outside world. In a set of 60 consecutive simultaneous displacements of two stripes, they were more likely to produce a response with the same polarity as the preceding one than a random polarity selection predicted. This suggested a dwelling of the FoA on one side of the visual field. Assuming that each response was influenced by the previous one in a way that the probability to repeat the response polarity was increased by a certain factor (dwelling factor, df), a random selection of response type including a df was computed. Implementation of the df removed the difference between observed probability of polarity repetition and the one suggested by random selection. When the interval between displacements was iteratively increased to 5s, no significant df could be detected anymore for pauses longer than 4s. In conclusion, Drosophila has an attention span of approximately 4s. Flies with a mutation in the radish gene expressed no after-effect of cueing and had a shortened attention span of about 1s. The dDAT inhibitor methylphenidate is able to rescue the first, but does not affect the latter phenotype. Probably, radish is differently involved in the two mechanisms. This study showed, that endogenous (covert) shifts of spatially selective visual attention in the fly Drosophila can be internally and externally guided. The variables determining the quality of a cue turned out to be multifaceted and a more systematic approach is needed for a better understanding of what property or feature of the cue changes the way it is evaluated by the fly. A first step has been made to demonstrate that SVA is a fundamental process and compromising it can influence the characteristics of other behaviors like walking. The existence of an attention span, the dependence of SVA on dopamine as well as the susceptibility to pharmacological manipulations, which in humans are used to treat respective diseases, point towards striking similarities between SVA in humans and Drosophila.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{LuiblneeHermann2014,
  author    = {Luibl [n{\´e}e Hermann], Christiane},
  title     = {The role of the neuropeptides NPF, sNPF, ITP and PDF in the circadian clock of Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93796},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis. Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock. In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats. We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD. It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity. Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication. Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Ljaschenko2013,
  author    = {Ljaschenko, Dmitrij},
  title     = {Hebbian plasticity at neuromuscular synapses of Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Synaptic plasticity determines the development of functional neural circuits. It is widely accepted as the mechanism behind learning and memory. Among different forms of synaptic plasticity, Hebbian plasticity describes an activity-induced change in synaptic strength, caused by correlated pre- and postsynaptic activity. Additionally, Hebbian plasticity is characterised by input specificity, which means it takes place only at synapses, which participate in activity. Because of its correlative nature, Hebbian plasticity suggests itself as a mechanism behind associative learning. Although it is commonly assumed that synaptic plasticity is closely linked to synaptic activity during development, the mechanistic understanding of this coupling is far from complete. In the present study channelrhodopsin-2 was used to evoke activity in vivo, at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation led to increased incorporation of GluR-IIA-type glutamate receptors into postsynaptic receptor fields, thus boosting postsynaptic sensitivity. This phenomenon is input-specific. Conversely, GluR-IIA was rapidly removed from synapses at which neurotransmitter release failed to evoke substantial postsynaptic depolarisation. This mechanism might be responsible to tame uncontrolled receptor field growth. Combining these results with developmental GluR-IIA dynamics leads to a comprehensive physiological concept, where Hebbian plasticity guides growth of postsynaptic receptor fields and sparse transmitter release stabilises receptor fields by preventing overgrowth. Additionally, a novel mechanism of retrograde signaling was discovered, where direct postsynaptic channelrhodopsin-2 based stimulation, without involvement of presynaptic neurotransmitter release, leads to presynaptic depression. This phenomenon is reminiscent of a known retrograde homeostatic mechanism, of inverted polarity, where neurotransmitter release is upregulated, upon reduction of postsynaptic sensitivity.},
  subject      = {Synapse},
  language  = {en}
}
@phdthesis{Kistenpfennig2012,
  author    = {Kistenpfennig, Christa},
  title     = {Rhodopsin 7 and Cryptochrome - circadian photoreception in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72209},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Many organisms evolved an endogenous clock to adapt to the daily environmental changes caused by the earth's rotation. Light is the primary time cue ("Zeitgeber") for entrainment of circadian clocks to the external 24-h day. In Drosophila, several visual pigments are known to mediate synchronization to light: The blue-light photopigment Cryptochrome (CRY) and six well-described rhodopsins (Rh1-Rh6). CRY is present in the majority of clock neurons as well as in the compound eyes, whereas the location of rhodopsins is restricted to the photoreceptive organs - the compound eyes, the ocelli and the HB-eyelets. CRY is thought to represent the key photoreceptor of Drosophila's circadian clock. Nevertheless, mutant flies lacking CRY (cry01) are able to synchronize their locomotor activity rhythms to light-dark (LD) cycles, but need significantly longer than wild-type flies. In this behavior, cry01 mutants strongly resemble mammalian species that do not possess any internal photoreceptors and perceive light information exclusively through their photoreceptive organs (eyes). Thus, a mammalian-like phase-shifting behavior would be expected in cry01 flies. We investigated this issue by monitoring a phase response curve (PRC) of cry01 and wild-type flies to 1-h light pulses of 1000 lux irradiance. Indeed, cry01 mutants produced a mammalian-similar so called type 1 PRC of comparatively low amplitude (< 25\% of wild-type) with phase delays to light pulses during the early subjective night and phase advances to light pulses during the late subjective night (~1 h each). Despite the predominant role of CRY, the visual system contributes to the light sensitivity of the fly's circadian clock, mainly around dawn and dusk. Furthermore, this phase shifting allows for the slow re-entrainment which we observed in cry01 mutants to 8-h phase delays of the LD 12 h:12 h cycle. However, cry01 also showed surprising differences in their shifting ability: First of all, their PRC was characterized by a second dead zone in the middle of the subjective night (ZT17-ZT19) in addition to the usual unresponsiveness during the subjective day. Second, in contrast to wild-type flies, cry01 mutants did not increase their shift of activity rhythms neither in response to longer stimuli nor to light pulses of higher irradiance. In contrast, both 6-h light pulses of 1000 lux and 1-h light pulses of 10,000 lux light intensity during the early subjective night even resulted in phase advances instead of the expected delays. Thus, CRY seems to be not only responsible for the high light sensitivity of the wild-type circadian clock, but is apparently also involved in integrating and processing light information. Rhodopsin 7 (Rh7) is a yet uncharacterized protein, but became a good photoreceptor candidate due to sequence similarities to the six known Drosophila Rhs. The second part of this thesis investigated the expression pattern of Rh7 and its possible functions, especially in circadian photoreception. Furthermore, we were interested in a potential interaction with CRY and thus, tested cry01 and rh70 cry01 mutants as well. Rh1 is the main visual pigment of the Drosophila compound eye and expressed in six out of eight photoreceptors cells (R1-R6) in each of the ~800 ommatidia. Motion vision depends exclusively on Rh1 function but, moreover, Rh1 plays an important structural role and assures proper photoreceptor cell development and maintenance. In order to investigate its possible photoreceptive function, we expressed Rh7 in place of Rh1. Rh7 was indeed able to overtake the role of Rh1 in both aspects: It prevented retinal degeneration and mediated the optomotor response (OR), a motion vision-dependent behavior. At the transcriptional level, rh7 is expressed at approximately equal amounts in adult fly brains and retinas. Due to a reduced specificity of anti-Rh7 antibodies, we could not verify this result at the protein level. However, analysis of rh7 null mutants (rh70) suggested different Rh7 functions in vivo. Previous experiments strongly indicated an increased sensitivity of the compound eyes in the absence of Rh7 and suggested impaired light adaptation. We aimed to test this hypothesis at the levels of circadian photoreception. Locomotor activity rhythms are a reliable output of the circadian clock. Rh70 mutant flies generally displayed a wild-type similar bimodal activity pattern comprising morning (M) and evening (E) activity bouts. Activity monitoring supported the proposed "shielding" function, since rh70 mutants behaved like wild-type flies experiencing high irradiances. Under all investigated conditions, their activity peaks lay further apart resulting in a prolonged midday break. The behavior of cry01 mutants was mainly characterized by an unexpectedly high flexibility in the timing of M and E activity bouts which allowed tracking of lights-on and lights-off even under extreme photoperiods. Activity profiles of the corresponding rh70 cry01 double mutants reflected neither synergistic nor antagonistic effects of Rh7 and CRY and were dominated by a broad E activity peak. In the future, the different circadian phenotypes will be further investigated on the molecular level by analysis of clock protein cycling in the underlying pacemaker neurons. The work of this thesis confirmed that Rh7 is indeed able to work as a photoreceptor and to initiate the classical phototransduction cascade. On the other hand, it provided further evidence at the levels of circadian photoreception that Rh7 might serve as a shielding pigment for Rh1 in vivo, thereby mediating proper light adaptation.},
  language  = {en}
}
@phdthesis{Cook2012,
  author    = {Cook, Mandy},
  title     = {The neurodegenerative Drosophila melanogaster AMPK mutant loechrig},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72027},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {In dieser Doktorarbeit wird die Drosophila Mutante loechrig (loe), die progressive Degeneration des Nervensystems aufweist, weiter beschrieben. In der loe Mutante fehlt eine neuronale Isoform der γ- Untereinheit der Proteinkinase AMPK (AMP-activated protein kinase). Die heterotrimere AMPK (auch als SNF4Aγ bekannt) kontrolliert das Energieniveau der Zelle, was st{\"a}ndiges Beobachten des ATP/AMP- Verh{\"a}ltnis erfordert. AMPK wird durch niedrige Energiekonzentrationen und Beeintr{\"a}chtigungen im Metabolismus, wie zum Beispiel Sauerstoffmangel, aktiviert und reguliert mehrere wichtige Signaltransduktionswege, die den Zellmetabolismus kontrollieren. Jedoch ist die Rolle von AMPK im neuronalen {\"U}berleben noch unklar. Eines der Proteine, dass von AMPK reguliert wird, ist HMGR (hydroxymethylglutaryl-CoA- reductase), ein Schl{\"u}sselenzym in der Cholesterin- und Isoprenoidsynthese. Es wurde gezeigt, dass wenn die Konzentration von HMGR manipuliert wird, auch der Schweregrad des neurodegenerativen Ph{\"a}notyps in loe beeinflusst wird. Obwohl die regulatorische Rolle von AMPK auf HMGR in Drosophila konserviert ist, k{\"o}nnen Insekten Cholesterin nicht de novo synthetisieren. Dennoch ist der Syntheseweg von Isoprenoiden zwischen Vertebraten und Insekten evolution{\"a}r konserviert. Isoprenylierung von Proteinen, wie zum Beispiel von kleinen G-Proteinen, stellt den Proteinen einen hydophobischen Anker bereit, mit denen sie sich an die Zellmembran binden k{\"o}nnen, was in anschließender Aktivierung resultieren kann. In dieser Doktorarbeit wird gezeigt, dass die loe Mutation die Prenylierung von Rho1 und den LIM-Kinasesignalweg beeinflusst, was eine wichtige Rolle im Umsatz von Aktin und axonalem Auswachsen spielt. Die Ergebnisse weisen darauf hin, dass die Mutation in LOE, Hyperaktivit{\"a}t des Isoprenoidsynthesewegs verursacht, was zur erh{\"o}hten Farnesylierung von Rho1 und einer dementsprechend h{\"o}heren Konzentration von Phospho- Cofilin f{\"u}hrt. Eine Mutation in Rho1 verbessert den neurodegenerativen Ph{\"a}notyp und die Lebenserwartung von loe. Der Anstieg vom inaktiven Cofilin in loe f{\"u}hrt zu einer Zunahme von filament{\"o}sen Aktin. Aktin ist am Auswachen von Neuronen beteiligt und Experimente in denen loe Neurone analysiert wurden, gaben wertvolle Einblicke in eine m{\"o}gliche Rolle die AMPK, und dementsprechend Aktin, im Neuronenwachstum spielt. Des Weiteren wurde demonstriert, dass Neurone, die von der loe Mutante stamen, einen verlangsamten axonalen Transport aufweisen, was darauf hinweist dass Ver{\"a}nderungen, die durch den Einfluss von loe auf den Rho1 Signalweg im Zytoskelettnetzwerk hervorgerufen wurden, zur St{\"o}rung des axonalen Transports und anschließenden neuronalen Tod f{\"u}hren. Es zeigte außerdem, dass Aktin nicht nur am neuronalen Auswachsen beteiligt ist, sondern auch wichtig f{\"u}r die Aufrechterhaltung von Neuronen ist. Das bedeutet, dass {\"A}nderungen der Aktindynamik zur progressiven Degeneration von Neuronen f{\"u}hren kann. Zusammenfassend unterstreichen diese Ergebnisse die wichtige Bedeutung von AMPK in den Funktionen und im {\"U}berleben von Neuronen und er{\"o}ffnen einen neuartigen funktionellen Mechanismus in dem {\"A}nderungen in AMPK neuronale Degeneration hervorrufen kann.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Chen2012,
  author    = {Chen, Yi-chun},
  title     = {Experimental access to the content of an olfactory memory trace in larval Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83705},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order "cortical" brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Schleyer2012,
  author    = {Schleyer, Michael},
  title     = {Integrating past, present and future: mechanisms of a simple decision in larval Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78923},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The 'decision' whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food - being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment - being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the 'decision' process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.},
  subject      = {Lernen},
  language  = {en}
}