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Die vier Crz-Neurone des ventralen Nervensystems von Drosophila melanogaster sammeln Evidenz, wann im Rahmen eines Paarungsakts zirka 6 Minuten vergangen sind. Diese Entscheidung ist für die männliche Fliege von Bedeutung, da das Männchen vor Ablauf dieser ~6 Minuten, welche den Zeitpunkt der Ejakulation darstellen, eher das eigene Leben opfern würde, als dass es die Paarung beenden würde. Nach Ablauf der ~6 Minuten fällt die Motivation des Männchens dagegen dramatisch ab. Im Rahmen der vorliegenden Arbeit wurde zunächst mittels optogenetischer neuronaler Inhibitionsprotokolle sowie Verhaltensanalysen das Phänomen der Evidenz-akkumulation in den Crz-Neuronen genauer charakterisiert. Dabei zeigte sich, dass die akkumulierte Evidenz auch während einer elektrischen Inhibition der Crz-Neurone persistierte. Dieses Ergebnis warf die Hypothese auf, dass das Äquivalent der akkumulierten Evidenz in den Crz-Neuronen biochemischer Natur sein könnte. Es wurde daraufhin ein Hochdurchsatzscreening-Verfahren entwickelt, mittels dessen 1388 genetische Manipulationen der Crz-Neurone durchgeführt und auf eine Änderung der Evidenzakkumulation getestet wurden. Nur ~30 genetische Manipulationen zeigten eine veränderte Evidenzakkumulation, wobei die meisten dieser Manipulationen den cAMP-Signalweg betrafen. Mittels der optogenetischen Photoadenylatzyklase bPAC, einer Reihe weiterer genetischer Manipulationen des cAMP-Signalwegs sowie der ex vivo Kalzium-Bildgebung und Fluoreszenzlebensdauer-Mikroskopie konnte bestätigt werden, dass cAMP das Äquivalent der in den Crz-Neuronen spannungsabhängig akkumulierten Evidenz darstellt, wobei die Kombination dieser Methoden nahelegte, dass der Schwellenwert der Evidenzakkumulation durch die cAMP-Bindungsaffinität der regulatorischen PKA-Untereinheiten festgelegt sein könnte. Mittels genetischer Mosaikexperimente sowie bildgebenden Verfahren konnte darüber hinaus gezeigt werden, dass innerhalb des Crz-Netzwerks eine positive Rückkopplungsschleife aus rekurrenter Aktivität sowie der cAMP-Akkumulation besteht, welche, sobald die cAMP-Spiegel den Schwellenwert erreichen, zu einem netzwerkweit synchronisierten massiven Kalziumeinstrom führt, was die Abgabe des Crz-Signals an nachgeschaltete Netzwerke triggert. Dieses Phänomen könnte ein Analogon des Aktionspotenzials auf Netzwerkebene sowie auf Intervallzeitskalen darstellen und wurde als „Eruption“ bezeichnet. Genetische, optogenetische sowie Bildgebungsexperimente konnten zeigen, dass die CaMKII derartige Eruptionen durch Niedrighalten der cAMP-Spiegel unterdrückt, was den Zeitmessmechanismus des ersten beschriebenen Intervallzeitmessers CaMKII offenlegt.
In dieser Arbeit wurden zwei Techniken zur Analyse der Funktion diverser Neuronen in Drosophila melanogaster angewendet. Im ersten Teil wurde mittels in-vivo Calcium Imaging Technik unter Verwendung des Calciumsensors Cameleon neuronale Aktivität entlang des olfaktorischen Signalweges registriert. Hierbei wurde die neuronale Repräsentation der Duftidentität und der Duftintensität untersucht. In Bezug auf diese Fragestellung wurde die Datenverarbeitung und Datenanalyse weiterentwickelt und standardisiert. Die Experimente führten zu dem Ergebnis, dass duftspezifische Aktivitätsmuster auf der Ebene des Antennallobus sehr gut unterscheidbar sind. Manche Aktivitätsmuster der präsentierten Düfte zeigten interessanterweise einen hohen Ähnlichkeitsgrad, wohingegen andere unähnlich waren. In höheren Gehirnzentren wie den Orten der terminalen Aborisationen der Projektionsneurone oder den Pilzkörper Kenyonzellen liegt eine starke Variabilität der duftevozierten Aktivitätsmuster vor, was generelle Interpretationen unmöglich macht und höchstens Vergleiche innerhalb eines Individuums zulässt. Des Weiteren konnte gezeigt werden, dass die Calciumsignale in den Rezeptorneuronen sowie prä- und postsynaptisch in den Projektionsneuronen bei Erhöhung der Konzentration der verschiedenen präsentierten Düfte über einen Bereich von mindestens drei Größenordnungen ansteigen. In den Kenyonzellen des Pilzkörper-Calyx und der Pilzkörper-Loben ist diese Konzentrationsabhängigkeit weniger deutlich ausgeprägt und im Falle der Loben nur für bestimmte Düfte detektierbar. Eine Bestätigung des postulierten „sparsed code“ der Duftpräsentation in den Pilzkörpern konnte in dieser Arbeit nicht erbracht werden, was möglicherweise daran liegt, dass eine Einzelzellauflösung mit der verwendeten Technik nicht erreicht werden kann. Im zweiten Teil dieser Arbeit sollte durch die Nutzung des lichtabhängigen Kationenkanals Channelrhodopsin-2 der Frage nachgegangen werden, ob bestimmte modulatorische Neurone die verstärkenden Eigenschaften eines bestrafenden oder belohnenden Stimulus vermitteln. Die lichtinduzierte Aktivierung von Channelrhodopsin-2 exprimierenden dopaminergen Neuronen als Ersatz für einen aversiven Reiz führte bei einer olfaktorischen Konditionierung bei Larven zur Bildung eines aversiven assoziativen Gedächtnisses. Im Gegensatz dazu induzierte die Aktivierung von Channelrhodopsin-2 in oktopaminergen/tyraminergen Neuronen als Ersatz für einen appetitiven Reiz ein appetitives assoziatives Gedächtnis. Diese Ergebnisse zeigen, dass dopaminerge Neurone bei Larven aversives Duftlernen, oktopaminerge/tyraminerge Neurone dagegen appetitives Duftlernen induzieren.
In this thesis, I introduce the Virtual Brain Protocol, which facilitates applications of the Standard Brain of Drosophila melanogaster. By providing reliable and extensible tools for the handling of neuroanatomical data, this protocol simplifies and organizes the recurring tasks involved in these applications. It is demonstrated that this protocol can also be used to generate average brains, i.e. to combine recordings of several brains with the same features such that the common features are emphasized. One of the most important steps of the Virtual Insect Protocol is the aligning of newly recorded data sets with the Standard Brain. After presenting methods commonly applied in a biological or medical context to align two different recordings, it is evaluated to what extent this alignment can be automated. To that end, existing Image Processing techniques are assessed. I demonstrate that these techniques do not satisfy the requirements needed to guarantee sensible alignments between two brains. Then, I analyze what needs to be taken into account in order to formulate an algorithm which satisfies the needs of the protocol. In the last chapter, I derive such an algorithm using methods from Information Theory, which bases the technique on a solid mathematical foundation. I show how Bayesian Inference can be applied to enhance the results further. It is demonstrated that this approach yields good results on very noisy images, detecting apparent boundaries between structures. The same approach can be extended to take additional knowledge into account, e.g. the relative position of the anatomical structures and their shape. It is shown how this extension can be utilized to segment a newly recorded brain automatically.
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.
In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
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.
An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. 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 olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade.
In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick & Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well.
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.
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.
The development of ethanol tolerance is due to changes in synaptic plasticity. Since the mechanisms mediating synaptic plasticity are probably defective in the mutant hangAE10, it was a goal of the present study to find out how HANG contributes to synaptic plasticity. In particular, it was important to clarify in which neuronal process HANG plays a role. Antibody stainings against HANG revealed that the protein is localized in all neuronal nuclei of larval and adult brains; the staining is absent in hangAE10, thus confirming that this P-element insertion stock is a protein null for HANG. Detailed analysis of the subnuclear distribution of HANG showed that HANG immunoreactivity is enriched at distinct spots in the nucleus in a speckled pattern; these speckles are found at the inside of the nuclear membrane and do not colocalize with chromatin nor with the nucleolus; thus, HANG is probably involved in the stabilization, processing or export of RNAs. As synaptic plasticity can be studied in single neurons at the larval neuromuscular junction, the morphology of the synaptic terminals of hangAE10 mutants was analyzed at muscle 6/7, segment A4. These studies revealed that hangAE10 mutants display a 40 % increase in bouton number and axonal branch length; in addition, some boutons have an abnormal hourglass-like shape, suggesting that they are arrested in a semi-separated state following the initiation of bouton division. The increase in bouton number of hang mutants is mainly due to an increase in numbers of type Ib boutons. The analysis of the distribution of several synaptic markers in hang mutants did not show abnormalities. The presynaptic expression of HANG in hang mutants rescues the increase in bouton number and axonal branch length, thus proving that the phenotypes seen in the P-element insertion hangAE10 are attributable to the lack of HANG rather than to effects of the P-element marker rosy or to a secondary hit on the same chromsome during mutagensis. This finding is further supported by the fact that postsynaptic expression of HANG does not rescue the abnormal NMJ morphology of hangAE10. Alterations in cAMP levels regulate the number of boutons; since hang mutants display an increase in bouton number, the questions was whether this morphological abnormality was due to defects in cAMP signalling. To test this hypothesis, hangAE10 NMJs were compared to those of the hypomorphic allele dnc1 that has a defective cAMP cascade. Some aspects of the NMJ phenotype (e.g. the increase in bouton number and the unaltered ratio of active zones per bouton area) are similar in hangAE10 and dnc1, other differ. Expression of a UAS-dnc transgene in hangAE10 mutants does not modify the phenotype. In summary, the results of this study indicate that nuclear protein HANG might be involved in isoform-specific splicing of genes required for synaptic plasticity at the NMJ.
Die Messung der räumlich aufgelösten Aktivität von neuronalen Zellverbänden ist ein wichtiges Werkzeug, um die Funktionsweise von Gehirnen zu verstehen. Für diese Arbeit diente die Fruchtfliege Drosophila melanogaster mit ihrer gut beschriebenen Genetik und Neurobiologie als Untersuchungsobjekt. Bei der vorgelegten Arbeit lag eine zweigeteilte Aufgabenstellung vor: Zum einen wurde die Technik des in – vivo Calcium – Imagings mit Hilfe des genetisch codierten Sensors Yellow Cameleon 2.1 am Lehrstuhl komplett neu etabliert, zum anderen wurde mit der neuen Technik das Zusammenspiel der funktionellen Elemente neuronaler Systeme anhand der Fliegenolfaktorik untersucht. Sowohl die Experimente zur Depolarisation durch KCl, als auch die Experimente zur olfaktorischen Codierung, wurden mit dem Calciumsensor Yellow Cameleon 2.1 durchgeführt. Es wurde ausgehend von der Vorgängerversion Yellow Cameleon 2.0 durch gezielte Mutagenese von Sören Diegelmann erstellt. Eine Photomultiplier – basierte in – vitro Funktionsanalyse des rekombinanten Sensorproteins ergab eine Zunahme der Ratio EYFP / ECFP mit steigender Calciumkonzentration. Dabei konnte auch der ratiometrische FRET – Effekt des Cameleons verdeutlicht werden: Mit steigender Calciumkonzentration verschiebt sich das Verhältnis von EYFP – Fluoreszenz zu ECFP – Fluoreszenz zu höheren Ratiowerten. Durch Zugabe des Calciumchelators EGTA konnte außerdem die reversible Arbeitsweise des Sensors nachgewiesen werden. Das in die Fliege eingebrachte Yellow Cameleon 2.1 – Konstrukt wurde mittels der GAL4 – UAS – Technik in verschiedenen olfaktorischen Gehirnzentren exprimiert. Von besonderer Relevanz für die Experimente zur olfaktorischen Codierung war dabei die GAL4 – Treiberlinie GH146. Mit ihrer Hilfe konnte das Fusionsprotein in den olfaktorischen Projektionsneuronen des Fliegengehirns exprimiert, und so die Duftrepräsentation im postsynaptischen Neuropil der Antennalloben bzw. in den präsynaptischen Neuropilen der Calyces und des lateralen Protocerbrums untersucht werden: Die Stimulation von 3 individuellen Fliegen mit den Düften Benzaldehyd, Isoamylacetat und Octanol liefert duftspezifische neuronale Aktivitätsmuster im Antenallobus. Die auf die Duftstimuli mit Calciumsignalen reagierenden Areale haben eine Größe von 10 – 30 µm, liegen also in der Größenordnung von individuellen Glomeruli. Die Duftrepräsentation in den Antennalloben zeigt außerdem einen kombinatorischen Aspekt: Jeder Duft evoziert ein charakteristisches Aktivitätsmuster bestehend aus einem oder mehreren Glomeruli. Die Aktivitätsmuster verschiedener Düfte können sich überlagern, d.h. individuelle Glomeruli können durch verschiedene Düfte aktiviert werden, das gesamte Aktivitätsmuster, d.h. die Summe der aktivierten Glomeruli eines bestimmten Duftes, ist jedoch charakteristisch. Die Duftrepräsentation in den Antennalloben von Drososophila geschieht also in Form eines glomerulären Codes, ein Prinzip der Duftverarbeitung, das auch in anderen Insekten und Vertebraten nachgewiesen werden konnte. Für den Calyx des Pilzkörpers ergaben sich innerhalb eines Individuums, bei wiederholter Stimulation mit demselben Duft, ebenfalls duftspezifische Aktivitätsmuster. Dabei waren die auf den Duftstimulus hin antwortenden neuronalen Areale diskret über den Calyx hinweg verteilt. Insgesamt zeigt das hohe Maß an Reproduzierbarkeit der Aktivitätsmuster für einen gegebenen Duft, dass im Calyx, wie in den Antennalloben, eine duftspezifische räumliche Repräsentation vorliegt. Der kombinatorische Aspekt der Codierung konnte auch hier beobachtet werden. Die einzelnen Spots der im Calyx gemessenen Aktivitätsmuster liegen in der Größenordnung von 5 +/- 2 µm und entsprechen somit in ihrer Größe den elektronenmikroskopisch beschriebenen Microglomeruli. Durch die Calcium – Imaging Experimente am lateralen Protocerebrum konnte nachgewiesen werden, dass die Erhöhung der Duftkonzentration eine räumliche Ausdehnung des aktivierten Neuropils zur Folge hat. Die EYFP –, ECFP – und Ratio – Intensitäten, die aus einer “Region of Interest“ im anterioren Bereich des lateralen Protocerebrums berechnet wurden, zeigen weiterhin, dass mit steigender Duftkonzentration auch die Stärke des Calciumsignals zunimmt. Dabei gibt es zwischen den 4 getesteten Düften statistisch signifikante Unterschiede: Methylcyclohexanol evoziert über den gesamten Verdünnungsbereich hinweg die schwächste neuronale Aktivität, Isoamylacetat evoziert in den Verdünnungsstufen 10-3 und 10-1 die stärkste neuronale Aktivität. D.h. neben der räumlichen Ausdehnung des Signals, führt die Konzentrationserhöhung auch zu einer gesteigerten Intensität des Calciumsignals, wobei sich die Signalintensitäten für verschiedene Düfte und Verdünnungsstufen unterscheiden können. Mit der verwendeten Versuchsanordnung und Datenauswertung, war es jedoch bislang nicht möglich eine räumliche Repräsentation der Düfte im lateralen Protocerebrum nachzuweisen.
Eine der größten Herausforderungen in der Neurobiologie ist es, die neuronalen Prozesse zu verstehen, die Lernen und Gedächtnis zugrundeliegen. Welche biochemischen Pfade liegen z.B. der Koinzidenzdetektion von Reizen (klassische Konditionierung) oder einer Handlung und ihren Konsequenzen (operante Konditionierung) zugrunde? In welchen neuronalen Unterstrukturen werden diese Informationen gespeichert? Wie ähnlich sind die Stoffwechselwege, die diese beiden Arten des assoziativen Lernens vermitteln und auf welchem Niveau divergieren sie? Drosophila melanogaster ist wegen der Verfügbarkeit von Lern-Paradigmen und neurogenetischen Werkzeugen ein geeigneter Modell-Organismus, zum diese Fragen zu adressieren. Er ermöglicht eine umfangreiche Studie der Funktion des Gens S6KII, das in der Taufliege in klassischer und operanter Konditionierung unterschiedlich involviert ist (Bertolucci, 2002; Putz et al., 2004). Rettungsexperimenten zeigen, dass die olfaktorische Konditionierung in der Tully Maschine (ein klassisches, Pawlow’sches Konditionierungsparadigma) von dem Vorhandensein eines intakten S6KII Gens abhängt. Die Rettung war sowohl mit einer vollständigen, als auch einer partiellen Deletion erfolgreich und dies zeigt, dass der Verlust der phosphorylierenden Untereinheit der Kinase die Hauptursache des Funktionsdefektes war. Das GAL4/UAS System wurde benutzt, um die S6KII Expression zeitlich und räumlich zu steuern. Es wurde gezeigt, dass die Expression der Kinase während des adulten Stadiums für die Rettung hinreichend war. Dieser Befund schließt eine Entwicklungsstörung als Ursache für den mutanten Phänotyp aus. Außerdem zeigte die gezielte räumliche Rettung von S6KII die Notwendigkeit der Pilzkörper und schloss Strukturen wie das mediane Bündel, die Antennalloben und den Zentralkomplex aus. Dieses Muster ist dem vorher mit der rutabaga Mutation identifizierten sehr ähnlich (Zars et al., 2000). Experimente mit der Doppelmutante rut, ign58-1 deuten an, dass rutabaga und S6KII im gleichen Signalweg aktiv sind. Vorhergehende Studien hatten bereits gezeigt, dass die unterschiedlichen Ergebnisse bei operanter und klassischer Konditionierung auf verschiedenen Rollen für S6KII in den zwei Arten des Lernens hindeuten (Bertolucci, 2002; Putz, 2002). Diese Schlussfolgerung wurde durch den mutanten Phänotyp der transgenen Linien in der Positionskonditionierung und ihr wildtypisches Verhalten in der klassischen Konditionierung zusätzlich bekräftigt. Eine neue Art von Lern-Experiment, genannt „Idle Experiment“, wurde entworfen. Es basiert auf der Konditionierung der Laufaktivität, stellt eine operante Aufgabenstellung dar und überwindet einige der Limitationen des „Standard“ Heat-Box Experimentes. Die neue Art des Idle Experimentes erlaubt es, „gelernte Hilflosigkeit“ in Fliegen zu erforschen, dabei zeigte sich eine erstaunliche Ähnlichkeit zu den Vorgängen in komplizierteren Organismen wie Ratten, Mäusen oder Menschen. Gelernte Hilflosigkeit in der Taufliege wurde nur in den Weibchen beobachtet und wird von Antidepressiva beeinflusst.
OMB and ORG-1
(2002)
Members of the T-box gene family encode transcription factors that play key roles during embryonic development and organogenesis of invertebrates and vertebrates. The defining feature of T-box proteins is an about 200 aa large, conserved DNA binding motif, the T domain. Their importance for proper development is highlighted by the dramatic phenotypes of T-box mutant animals. My thesis was mainly focused on two Drosophila T-box genes, optomotor-blind (omb) and optomotor-blind related 1 (org-1), and included (i) a genetic analysis of org-1 and (ii) the identification of molecular determinants within OMB and ORG-1 that confer functional specificity. (i) Genetic analysis of org-1 initially based on a behavioral Drosophila mutant, C31. C31 is a X-linked, recessive mutant and was mapped to 7E-F, the cytological region of org-1. This pleiotropic mutant is manifested in walking defects, structural aberrations in the central brain, and "held-out" wings. Molecular analysis revealed that C31 contains an insertion of a 5' truncated I retrotransposon within the 3' untranslated transcript of org-1, suggesting that C31 might represent the first org-1 mutant. Based on this hypothesis, we screened 44.500 F1 female offspring of EMS mutagenized males and C31 females for the "held-out" phenotype, but failed to isolate any C31 or org-1 mutant, although this mutagenesis was functional per se. Since we could not exclude the possibility that our failure is due to an idiosyncracy of C31, we intended not to rely on C31 in further genetic experiments and followed a reverse genetic strategy . All P element lines cytologically mapping to 7E-7F were characterized for their precise insertion sites. 13 of the 19 analyzed lines had P element insertions within a hot-spot 37 kb downstream of org-1. No P element insertions within org-1 could be identified, but several P element insertions were determined on either side of org-1. The org-1 nearest insertions were used for local-hop experiments, in which we associated 6 new genes with P insertions, but failed to target org-1. The closest P elements are still 10 kb away from org-1. Subsequently, we employed org-1 flanking P elements to induce precise deletions in 7E-F spanning org-1. Two org-1 flanking P elements were brought together on a recombinant chromosome. Remobilization of P elements in cis configuration frequently results in deletions with the P element insertion sites as deficiency endpoints. In a first attempt, we expected to identify deficiencies by screening for C31 alleles. 8 new C31 alleles could be isolated. The new C31 chromosomes, however, did not carry the desired deletion. Molecular analysis indicated that C31 is not caused by aberrations in org-1, but by mutations in a distal locus. We repeated the P element remobilization and screened for the absence of P element markers. 4 lethal chromosomes could be isolated with a deletion of the org-1 locus. (ii) The consequences of ectopic org-1 were analyzed using UAS-org-1 transgenic flies and a number of different Gal4 driver lines. Misexpression of org-1 during imaginal development interfered with the normal development of many organs and resulted in flies with a plethora of phenotypes. These include a homeotic transformation of distal antenna (flagellum) into distal leg structures, a strong size reduction of the legs along their proximo-distal axis, and stunted wings. Like ectopic org-1, ectopic omb leads to dramatic changes of normal developmental pathways in Drosophila as well. dpp-Gal4/ UAS-omb flies are late pupal lethal and show an ectopic pair of wings and largely reduced eyes. GMR-Gal4 driven ectopic omb expression in the developing eye causes a degeneration of the photoreceptor cells, while GMR-Gal4/ UAS-org-1 flies have intact eyes. Hence, ectopic org-1 and omb induce profound phenotypes that are qualitatively different for these homologous genes. To begin to address the question where within OMB and ORG-1 the specificity determinants reside, we conceptionally subdivided both proteins into three domains and tested the relevance ofthese domains for functional specificity in vivo. The single domains were cloned and used as modules to assemble all possible omb-org-1 chimeric trans- genes. A method was developed to determine the relative expression strength of different UAS-transgenes, allowing to compare the various transgenic constructs for qualitative differences only, excluding different transgene quantities. Analysis of chimeric omb-org-1 transgenes with the GMR-Gal4 driver revealed that all three OMB domains contribute to functional specificity.
It has been known for a long time that Drosophila can learn to discriminate not only between different odorants but also between different concentrations of the same odor. Olfactory associative learning has been described as a pairing between odorant and electric shock and since then, most of the experiments conducted in this respect have largely neglected the dual properties of odors: quality and intensity. For odorant-coupled short-term memory, a biochemical model has been proposed that mainly relies on the known cAMP signaling pathway. Mushroom bodies (MB) have been shown to be necessary and sufficient for this type of memory, and the MB-model of odor learning and short-term memory was established. Yet, theoretically, based on the MB-model, flies should not be able to learn concentrations if trained to the lower of the two concentrations in the test. In this thesis, I investigate the role of concentration-dependent learning, establishment of a concentration-dependent memory and their correlation to the standard two-odor learning as described by the MB-model. In order to highlight the difference between learning of quality and learning of intensity of the same odor I have tried to characterize the nature of the stimulus that is actually learned by the flies, leading to the conclusion that during the training flies learn all possible cues that are presented at the time. The type of the following test seems to govern the usage of the information available. This revealed a distinction between what flies learned and what is actually measured. Furthermore, I have shown that learning of concentration is associative and that it is symmetrical between high and low concentrations. I have also shown how the subjective quality perception of an odor changes with changing intensity, suggesting that one odor can have more than one scent. There is no proof that flies perceive a range of concentrations of one odorant as one (odor) quality. Flies display a certain level of concentration invariance that is limited and related to the particular concentration. Learning of concentration is relevant only to a limited range of concentrations within the boundaries of concentration invariance. Moreover, under certain conditions, two chemically distinct odorants could smell sufficiently similarly such, that they can be generalized between each other like if they would be of the same quality. Therefore, the abilities of the fly to identify the difference in quality or in intensity of the stimuli need to be distinguished. The way how the stimulus is analyzed and processed speaks in favor of a concept postulating the existence of two separated memories. To follow this concept, I have proposed a new form of memory called odor intensity memory (OIM), characterized it and compared it to other olfactory memories. OIM is independent of some members of the known cAMP signaling pathway and very likely forms the rutabaga-independent component of the standard two-odor memory. The rutabaga-dependent odor memory requires qualitatively different olfactory stimuli. OIM is revealed within the limits of concentration invariance where the memory test gives only sub-optimal performance for the concentration differences but discrimination of odor quality is not possible at all. Based on the available experimental tools, OIM seems to require the mushroom bodies the same as odor-quality memory but its properties are different. Flies can memorize the quality of several odorants at a given time but a newly formed memory of one odor interferes with the OIM stored before. In addition, the OIM lasts only 1 to 3 hours - much shorter than the odor-quality memory.
Aggression is a strikingly multi-faceted phenomenon occurring in vertebrates as well as in invertebrates. Despite its omnipresence, the neuronal basis of aggressive behaviours is yet barely understood. Many studies however, imply a role for biogenic amines in aggression. This PhD project aimed at contributing to the understanding of the neuronal correlates of aggression, with a main focus on the biogenic amine octopamine, using Drosophila melanogaster as the model system. In Drosophila, agonistic encounters of males and females are composed of a variety of both offensive and defensive components, some of which are displayed more often in one sex than in the other. To simplify analysis and to standardize evaluation, I chose to focus on a single indicator of aggression: the lunge, a striking feature unique to Drosophila male aggression. By evaluating the lunge I developed in cooperation with Andreas Eckart for the first time an automated, video-based analysis of Drosophila male aggression. The present software program gives the number of lunges for each fly in a certain time interval. In addition, it provides information such as the distance the fly walked and his size among others. In combination with a second software program that we developed, aggressive interactions between two male Drosophila melanogaster of a genotype of choice can now be registered either completely automatically or if preferred semi-automatically. Using these softwares, I demonstrate that (1) body size differences of 8% and higher influence the outcome of a fight in favour of the larger male; (2) walking activity alters lunge frequency with more lunges performed by more active pairs of males; (3) flies mutant for the white gene, one member of the ABC transporter family in Drosophila, are profoundly impaired in aggression, an effect that is partially due to reduced visual performance. (4) Either knocking-down white in various brain regions or chemically ablating the mushroom body located in the central brain by deleting its neuroblast precursors diminishes aggression, indicating that integrity of various neural circuits/brain regions is required for wild-type aggression to occur. Furthermore, I show that (5) flies lacking octopamine signalling but having altered tyramine signalling display hardly any lunge. A quantitative high-speed analysis revealed that lunge execution is almost indistinguishable from wild-type males. The results from the experiments in which octopamine levels and/or tyramine levels were restored suggest that an elaborate pattern of octopamine levels in time and space is required to enable flies to express wild-type aggressive behaviour.
Morphologie und Organisation individueller oktopaminerger Neurone im Gehirn von Drosophila m.
(2009)
Das biogene Amin Oktopamin moduliert verschiedene Verhaltensweisen in Invertebraten. In verschiedenen Insektenspezies, wie Heuschrecken, Grillen oder Schaben, ist die Funktion und die Architektur des peripheren oktopaminergen Systems auf Einzelzellebene bekannt. Um die zelluläre Grundlage für die verschiedenen Funktionen von Oktopamin im Zentralnervensystem zu verstehen, ist eine detaillierte Analyse der Architektur des zentralen oktopaminergen Systems notwendig. Innerhalb meiner Doktorarbeit fertigte eine anatomische Karte individueller oktopaminerger Neurone des adulten Hirns von Drosophila an. Ich nutzte die Flp-out Technik, um einzelne oktopaminerge Neurone anzufärben. Anhand ihrer Projektionsmuster konnte ich 28 verschiedene Zelltypen in vier Oktopamin-immunoreaktiven Zellclustern identifizieren. Ihre Morphologie sowie die Verteilung genetischer Marker zeigte, dass die meisten Zelltypen mehrere Neuropile innervieren und dabei eine klare Trennung von Prä- und Postsynaptischen Regionen aufweisen. Die Mehrheit der Zelltypen bildet dendritische Verzweigungen in einer bestimmten Region, der posterioren Slope. Jedoch innerviert jeder Zelltyp stereotyp eine bestimmte Kombination von Zielregionen im Gehirn. Das deutet stark darauf hin, dass oktopaminerge Neurone kombinatorisch organisiert sind: Jedes individuelle Neuron scheint Komponente eines spezifischen neuronalen Schaltkreises zu sein. Dabei könnte jeder Zelltyp eine Art “Modul” darstellen, das selektiv bestimmte Funktionen in den jeweiligen Zielregionen moduliert. Das oktopaminerge Mittelliniencluster des Subösophagealen Ganglions zeigt eine besondere zelluläre Organisation. Es besteht aus gepaarten und ungepaarten Neuronen, die des Zentralgehirn mit extensiven Verzweigungen versorgen. Um die Ordnung hinter dieser komplexen Organisation zu verstehen, wurden die segmentale Organistion der Mittellinienneurone auf Einzelzellebene analysiert und ihre embryonalen Anlagen verglichen. Letzteres ermöglichte die morphologische Analyse von einzelnen oktopaminergen Mittellinienklonen. OA-VPM und OA-VUM Neurone bilden zusammen drei Subcluster im Subösophagealen Ganglion, die wahrscheinlich die drei gnathalen Neuromere repräsentieren. Alle OA-VUM Neurone stammen von der embryonalen Mittellinie ab. In den mandibularen und maxillaren Neuromeren formen sie morphologisch identische Zelltypen, mit stereotypen Innervationsmustern. OA-VPM Neurone gehen nicht aus der embryonalen Mittellinie hervor und sind nicht segmental dupliziert. Diese Arbeit vermittelt nicht nur einen Eindruck über die Architektur individueller oktopaminerger Neurone, sondern auch über die Organisation des oktopaminergen Systems auf Einzelzellebene.
Durch genaue Kartierung der Defizienzen in den Mutanten konnten bislang unbekannte regulatorische Elemente des Synapsin Gens identifiziert werden. Mit dieser Information sollte es möglich sein, einen Synapsin-„Rescue“ Vektor zu konstruieren, der nach Transformation in die Nullmutante den wildtypischen Phänotyp wiederherstellt. Beim Vergleich der im Rahmen des Berkeley Drosophila Genom Projekt veröffentlichten Sequenz des Synapsin Gens mit vor sieben Jahren publizierten Sequenzdaten fielen Diskrepanzen sowohl in der genomischen Sequenz als auch in der cDNA auf. Um zu klären, ob es sich hier um Artefakte, Polymorphismen oder systematische Modifikationen handelt, wurde der entsprechende Bereich von neun an verschiedenen Orten gefangenen Wildtypen genomisch und auf der cDNA Ebene amplifiziert und sequenziert. In allen Fällen wurde die genomische Sequenz des Genomprojekts verifiziert, so dass von einem Sequenzierfehler in der früheren Sequenz auszugehen ist. Als Folge ergibt sich eine Exon-Intron Struktur, bei der die Spleiß-Konsensussequenz (GT-AG Regel) im Intron 4 des Synapsins gewahrt bleibt. Dagegen bestätigten die RT-PCR Sequenzen die früheren cDNA-Daten, so dass ein A zu G Austausch zwischen der genomischen Sequenz und der cDNA des Proteins aufgedeckt wird. Dieser Austausch führt zu einer Veränderung der in allen bisher bekannten Synapsinen konservierten Zielsequenz der Proteinkinase CaMK I/ IV und PKA, was interessante Fragen zu seiner funktionellen Bedeutung aufwirft. Die Basensubstitution spricht für ein A-zu-I RNA-Editing auf der Ebene der Ribonukleinsäure. Dieser Vorgang wird durch das Enzym dADAR katalysiert und wurde bereits für verschiedene neuronale Proteine nachgewiesen. Die für die Reaktion benötigte doppelsträngige Sekundärstruktur der RNA kann durch die Sequenz der prä-mRNA des Synapsins gebildet werden. Die potentielle „Editing site Complementary Sequence“ (ECS) konnte im Intron 4 in einem Abstand von ca. 90 Basen stromabwärts der Editing-Stelle durch ein Computerprogramm ermittelt werden. Der A zu G Austausch wird in allen Laborwildtypen und allen neu etablierten Stämmen, sowie in verschiedenen Entwicklungsstadien beobachtet. Lediglich in einem cDNA-Gemisch aus Eiern, Embryonen und 1. Larven findet man neben der editierten auch die nicht-editierte Sequenz. Um in späteren Experimenten die Funktion der Phosphorylierung und die Auswirkung der mRNA Editierung ermitteln zu können wurden in einem weiteren Versuch die beiden Erkennungsstellen der PKA in der cDNA durch Mutationen modifiziert, so dass Phosphorylierungstests an den Konstrukten durchgeführt werden können. Zur phänotypischen Charakterisierung der Nullmutante wurde die Defizienz-Linie Syn97 durch extensive Rückkreuzung in den genetischen Hintergrund des Wildtyps CantonS eingebracht, der als Standard-Kontrollstamm für Verhaltensexperimente und insbesondere Lernversuche dient. Die Linie Syn97CS wurde im Rahmen einer Kooperation von Mitarbeitern des Lehrstuhls in verschiedenen Verhaltenstests und Lernparadigmen auf phänotypische Veränderungen überprüft. Dabei fanden sich mehrere Verhaltensunterschiede zum Wildtyp, die vermutlich auf geringfügigen Modifikationen in komplexen neuronalen Netzwerken beruhen. In operanten Lernparadigmen konnte ein Einfluss der Synapsin-Elimination auf den Lernerfolg detektiert werden. Dabei trat die Reduktion des Lernindex bereits im dritten Larvenstadien auf und setzte sich in der adulten Fliege fort. Der Einfluss des Fehlens des Synapsins auf Lernprozesse in Drosophila steht im Einklang mit Befunden aus Knock-out Mäusen für SynI + II. Im reduzierten Courtship Index der Syn97CS Männchen offenbart sich ein konkreter Hinweis auf eine verringerte Darwin’sche Fitness der Synapsin-Nullmutante. Die Gesamtheit der in der Synapsin-Nullmutante entdeckten Phänotypen könnte den hohen Konservierungsgrad des Proteins zwischen Vertebraten und Invertebraten erklären. In einem weiteren Teil-Projekt konnten Mutationen in die cDNA des Calciumsensor Cameleon 2.0 Proteins eingebracht werden, um so die verbesserte Version Cam 2.1 zu erhalten. Daraufhin wurden mehrere transgene UAS-Cam 2.1 Linien hergestellt, die bei der Kreuzung mit verfügbaren Gal4 Linien den Calciumsensor für eine Expression in definierten Neuronenpopulationen von Drosophila zugänglich machen. In weiterführenden Arbeiten konnte die Funktionalität des Fusionsproteins überprüft werden und somit die ersten Schritte hin zur Anwendung der in-vivo Calcium Imaging Methode am Lehrstuhl durchgeführt werden.
Neurodegenerative Erkrankungen des Menschen sind eines der Hauptfelder molekularer neurobiologischer Grundlagenforschung. Um generell molekulare, komplizierte Vorgänge in vivo untersuchen zu können, nutzt man seit geraumer Zeit Modellorganismen wie Caenorhabditis elegans oder Drosophila melanogaster. In der vorliegenden Arbeit wird die Drosophila-Neurodegenerationsmutante loe (löchrig) beschrieben, die als Modell für die Rolle des Cholesterinhaushalts im Bezug auf Neurodegeneration herangezogen werden kann. Die Fliegen dieser Mutante zeigen stark progressive, altersabhängige Degeneration von Neuronen, dabei unterlaufen diese Nervenzellen einen nekrotischenZelltod. Verantwortlich für diese Mutation ist die Insertion eines P-Elementes in einem Intron des Drosophila-g-5'-AMP-aktivierten Proteinkinase- (AMPK)-Gens. Die verschiedenen Spleißprodukte des loe Gens kodieren für die regulatorische g-Untereinheit des AMPK-Komplexes, der , aktiviert durch 5'AMP, energieintensive Prozesse negativ reguliert. Die Spleißform loeI ist durch die P-Element-Insertion betroffen, Anteile des P-Elementes werden in das loeI-Transkript hineingespleißt. Eine neuronale Expression von loeI im loe-Hintergrund führt zur Revertierung des loe-Phänotypes. Mit der Expression anderer Spleißformen kann dieser Effekt nicht erzielt werden. Das LOE I-Protein birgt in seinem N-Terminus eine Reihe möglicher Interaktionstellen mit anderen Proteinen, die den AMPK-Komplex in einen Kontext mit den Proteinen der APP (Amyloid Precursor Proteins) ?Familie stellen oder z. B. Interaktionen mit dem Cytoskelett herstellen können. Eine molekulare Interaktion mit NiPSNAP, einem Protein, dass vermutlich eine Rolle im Vesikelverkehr spielt, konnte nachgewiesen werden. Ein direktes humanes Homolog von LOE I ist nicht bekannt, wohlgleich es im Menschen drei AMPK-g-Untereinheiten gibt, von denen zwei ähnliche Funktionen übernehmen könnten wie LOE I. Die loe-Mutante interagiert genetisch mit der Mutante clb ? columbus, die einen Defekt im Gen der HMG-CoA-Reduktase trägt. Dieses Emzym ist das Schlüsselenzym der Cholesterinbiosynthese. Die Art der Interaktion belegt eine negative Regulierung der HMG-CoA-Reduktase durch die AMPK. So schwächt die clb-Mutation den neurodegenerativen loe-Phänotyp ab, eine Überexpression von clb verstärkt diesen. Eine Verminderung der Neurodegeneration kann auch mit Medikamenten erreicht werden: Statine, potente Hemmer der HMG-COA-Reduktase, reprimieren deutlich den loe-Phänotyp. In loe ist der Cholesterinester-Spiegel auf 40% abgesenkt. Eine weitere genetische Interaktion von loe konnte nachgewiesen werden: Die Mutante für das Drosophila-Homolog von APP (Appl) verstärkt den neurodegenerativen Phänotyp in loe stark, wogegen die Appl-Mutante selbst keine neurodegenerativen Defekte aufweist. Darüberhinaus zeigt die Doppelmutante Defekte, die keine der Einzelmutanten aufweist: Sterilität oder eine extrem kurze Lebensdauer von nur 3-4 Tagen. Diese Interaktion ließ sich auf molekularer Ebene charakterisieren. Die proteolytische Prozessierung von APPL durch Sekretasen ist in loe alteriert. In der vorliegenden Arbeit konnte gezeigt werden, dass durch die loe-Mutation die b-Sekretase aus Vertebraten (BACE) und eine bisher noch nicht beschriebene endogene Sekretase aus Drosophila negativ beeiflusst werden. Ein AMPK-Komplex mit LOE I als g-Untereinheit scheint über den Cholesterinester-Spiegel die Aktivität einer speziellen Untergruppe der Sekretasen zu beeinflussen. Die Missfunktion dieser Sekretasen ist ein kritischer Punkt in der Pathogenese der Alzheimer-Krankheit. Die loe-Mutation wirft neues Licht auf die bekannten Verbindungen zwischen Cholesterin-Stoffwechsel, Vesikelverkehr und Prozessierung von APP(L). Mit den großen Möglichkeiten, die die Drosophila-Genetik bietet, stellt diese neue Mutante ein weiteres Werkzeug zur Charakterisierung von Therapie-Ansätzen für die Alzheimer-Kankheit dar. Die vorliegende Arbeit belegt um ein weiteres Mal, dass Drosophila ein potentes Modellsystem zur Untersuchung humaner, neurodegenerativer Erkrankungen wie Chorea Huntington, Parkinson oder der Alzheimer Krankheit ist.
In this thesis two genes involved in causing neurodegenerative phenotypes in Drosophila are described. olk (omb-like), a futsch allele, is a micotubule associated protein (MAP) which is homologous to MAP1B and sws (swiss cheese) a serine esterase of yet unknown function within the nervous system. The lack of either one of these genes causes progressive neurodegeneration in two different ways. The sws mutant is characterized by general degeneration of the adult nervous system, glial hyperwrapping and neuronal apoptosis. Deletion of NTE (neuropathy target esterase), the SWS homolog in vertebrates, has been shown to cause a similar pattern of progressive neural degeneration in mice. NTE reacts with organophosphates causing axonal degeneration in humans. Inhibition of vertebrate NTE is insufficient to induce paralyzing axonal degeneration, a reaction called "aging reaction" is necessary for the disease to set in. It is hypothesized that a second "non-esterase" function of NTE is responsible for this phenomenon. The biological function of SWS within the nervous system is still unknown. To characterize the function of this protein several transgenic fly lines expressing different mutated forms of SWS were established. The controlled expression of altered SWS protein with the GAL4/UAS system allowed the analysis of isolated parts of the protein that were altered in the respective constructs. The characterization of a possible non-esterase function was of particular interest in these experiments. One previously described aberrant SWS construct lacking the first 80 amino acids (SWSΔ1-80) showed a deleterious, dominant effect when overexpressed and was used as a model for organophosphate (OP) intoxication. This construct retains part of its detrimental effect even without catalytically active serine esterase function. This strongly suggests that there is another characteristic to SWS that is not defined solely by its serine esterase activity. Experiments analyzing the lipid contents of sws mutant, wildtype (wt) and SWS overexpressing flies gave valuable insights into a possible biological function of SWS. Phosphatidylcholine, a major component of cell membranes, accumulates in sws mutants whereas it is depleted in SWS overexpressing flies. This suggests that SWS is involved in phosphatidylcholine regulation. The produced α-SWS antibody made it possible to study the intracellular localization of SWS. Images of double stainings with ER (endoplasmic reticulum) markers show that SWS is in great part localized to the ER. This is consistent with findings of SWS/ NTE localization in yeast and mouse cells. The olk mutant also shows progressive neurodegeneration but it is more localized to the olfactory system and mushroom bodies. Regarding specific cell types it seemed that specifically the projection neurons (PNs) are affected. A behavioral phenotype consisting of poor olfactory memory compared to wt is also observed even before histologically visible neurodegeneration sets in. Considering that the projection neurons connect the antennal lobes to the mushroom bodies, widely regarded as the "learning center", this impairment was expected. Three mutants where identified (olk1-3) by complementation analysis with the previously known futschN94 allele and sequencing of the coding sequence of olk1 revealed a nonsense mutation early in the protein. Consistent with the predicted function of Futsch as a microtubule associated protein (MAP), abnormalities are most likely due to a defective microtubule network and defects in axonal transport. In histological sections a modified cytoskeletal network is observed and western blots confirm a difference in the amount of tubulin present in the olk1 mutant versus the wt. The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton. Observation of transport processes in primary neural cultures derived from olk1 mutant flies also showed a reduction of mitochondrial transport. Interaction with the fragile X mental retardation gene (dfmr1) was observed with the olk mutant. A dfmr1/ olk1 double mutant shows an ameliorated phenotype compared to the olk1 single mutant. tau, another MAP gene, was also shown to be able to partially rescue the olk1 mutant.