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Most natural learning situations are of a complex nature and consist of a tight conjunction of the animal's behavior (B) with the perceived stimuli. According to the behavior of the animal in response to these stimuli, they are classified as being either biologically neutral (conditioned stimuli, CS) or important (unconditioned stimuli, US or reinforcer). A typical learning situation is thus identified by a three term contingency of B, CS and US. A functional characterization of the single associations during conditioning in such a three term contingency has so far hardly been possible. Therefore, the operational distinction between classical conditioning as a behavior-independent learning process (CS-US associations) and operant conditioning as essentially behavior-dependent learning (B-US associations) has proven very valuable. However, most learning experiments described so far have not been successful in fully separating operant from classical conditioning into single-association tasks. The Drosophila flight simulator in which the relevant behavior is a single motor variable (yaw torque), allows for the first time to completely separate the operant (B-US, B-CS) and the classical (CS-US) components of a complex learning situation and to examine their interactions. In this thesis the contributions of the single associations (CS-US, B-US and B-CS) to memory formation are studied. Moreover, for the first time a particularly prominent single association (CS-US) is characterized extensively in a three term contingency. A yoked control shows that classical (CS-US) pattern learning requires more training than operant pattern learning. Additionally, it can be demonstrated that an operantly trained stimulus can be successfully transferred from the behavior used during training to a new behavior in a subsequent test phase. This result shows unambiguously that during operant conditioning classical (CS-US) associations can be formed. In an extension to this insight, it emerges that such a classical association blocks the formation of an operant association, which would have been formed without the operant control of the learned stimuli. Instead the operant component seems to develop less markedly and is probably merged into a complex three-way association. This three-way association could either be implemented as a sequential B-CS-US or as a hierarchical (B-CS)-US association. The comparison of a simple classical (CS-US) with a composite operant (B, CS and US) learning situation and of a simple operant (B-US) with another composite operant (B, CS and US) learning situation, suggests a hierarchy of predictors of reinforcement. Operant behavior occurring during composite operant conditioning is hardly conditioned at all. The associability of classical stimuli that bear no relation to the behavior of the animal is of an intermediate value, as is operant behavior alone. Stimuli that are controlled by operant behavior accrue associative strength most easily. If several stimuli are available as potential predictors, again the question arises which CS-US associations are formed? A number of different studies in vertebrates yielded amazingly congruent results. These results inspired to examine and compare the properties of the CS-US association in a complex learning situation at the flight simulator with these vertebrate results. It is shown for the first time that Drosophila can learn compound stimuli and recall the individual components independently and in similar proportions. The attempt to obtain second-order conditioning with these stimuli, yielded a relatively small effect. In comparison with vertebrate data, blocking and sensory preconditioning experiments produced conforming as well as dissenting results. While no blocking could be found, a sound sensory preconditioning effect was obtained. Possible reasons for the failure to find blocking are discussed and further experiments are suggested. The sensory preconditioning effect found in this study is revealed using simultaneous stimulus presentation and depends on the amount of preconditioning. It is argued that this effect is a case of 'incidental learning', where two stimuli are associated without the need of reinforcement. Finally, the implications of the results obtained in this study for the general understanding of memory formation in complex learning situations are discussed.
Different transgenes that can be expressed in neurons to kill or block them were compared. Tetanus neurotoxin blocked chemical synapses very efficiently. Synapses consisting of a chemical and an electrical component were blocked more reliably by expressing a human inwardly rectifying potassium channel. To gain temporal control over neuronal function, three genetic tools have been investigated. None of the systems is without drawbacks, however, the recombination induced tetanus neurotoxin expression is a promising approach. The knowledge gained from the comparative methodological study was used to investigate the role of neurons in sensory systems in processing different sensory informations. Receptor neurons sensitive for chemical or mechanical stimuli were correlated to specific olfactory behaviors or locomotor tasks. The main topic of this thesis is the much discussed question of which neurons are involved in motion processing in the visual system of flies. Neither L2 nor L4 neurons in the first visual neuropil are essential for motion-detection. The results indicate that maybe motion is detected by the network of amacrine cells (a). The vertical motion-sensitive VS cells in the lobula plate are not necessary for behavioral responses to vertical motion. This finding implies that the lack of VS cells in the structural mutant optomotor blind is not causally related to the altered responses to motion stimuli. Other abnormalities in optomotor blind are responsible for this behavioral phenotype. This work shows the potential of the described methods in studying information processing in the Drosophila brain. Groups of neurons were correlated to complex behavioral responses and theories about information processing were tested by behavioral experiments with transgenic flies. The refinement of the genetic tools to interfere with neuronal function will make the Drosophila brain an even better model to study information processing in nervous systems.
Characterization of memories and ignorant (S6KII) mutants in operant conditioning in the heat-box
(2002)
Learning and memory processes of operant conditioning in the heat-box were analysed. Age, sex, and larval desity were not critical parameters influencing memory, while low or high activity levels of flies were negatively correlated with their performance. In a search for conditioning parameters leading to high retention scores, intermittent training was shown to give better results than continuous training. As the memory test is the immediate continuation of the conditioning phase just omitting reinforcement, we obtain a memory which consists of two components: a spatial preference for one side of the chamber and a stay-where-you-are effect in which the side preference is contaminated by the persistence of heat avoidance. Intermittent training strengthens the latter. In the next part, memory retention was investigated. Flies were trained in one chamber and tested in a second one after a brief reminder training. With this direct transfer, memory scores reflect an associative learning process in the first chamber. To investigate memory retention after extended time periods, indirect transfer experiments were performed. The fly was transferred to a different environment between training and test phases. With this procedure an after-effect of the training was still observed two hours later. Surprisingly, exposure to the chamber without conditioning also lead to a memory effect in the indirect transfer experiment. This exposure effect revealed a dispositional change that facilitates operant learning during the reminder training. The various memory effects are independent of the mushroom bodies. The transfer experiments and yoked controls proved that the heat-box records an associative memory. Even two hours after the operant conditioning procedure, the fly remembers that its position in the chamber controls temperature. The cAMP signaling cascade is involved in heat-box learning. Thus, amnesiac, rutabaga, and dunce mutants have an impaired learning / memory. Searching for, yet unknown, genes and signaling cascades involved in operant conditioning, a Drosophila melanogaster mutant screen with 1221 viable X-chromosome P-element lines was performed. 29 lines with consistently reduced heat avoidance/ learning or memory scores were isolated. Among those, three lines have the p[lacW] located in the amnesiac ORF, confirming that with the chosen candidate criteria the heat-box is a useful tool to screen for learning and /or memory mutants. The mutant line ignP1 (8522), which is defective in the gene encoding p90 ribosomal S6 kinase (S6KII), was investigated. The P-insertion of line ignP1 is the first Drosophila mutation in the ignorant (S6KII) gene. It has the transposon inserted in the first exon. Mutant males are characterized by low training performance, while females perform well in the standard experiment. Several deletion mutants of the ignorant gene have been generated. In precise jumpouts the phenotype was reverted. Imprecise jumpouts with a partial loss of the coding region were defective in operant conditioning. Surprisingly, null mutants showed wild-type behavior. This might indicate an indirect effect of the mutated ignorant gene on learning processes. In classical odor avoidance conditioning, ignorant null mutants showed a defect in the 3-min, 30-min, and 3-hr memory, while the precise jumpout of the transposon resulted in a reversion of the behavioral phenotype. Deviating results from operant and classical conditioning indicate different roles for S6KII in the two types of learning.
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.
In the last years it became evident that many cytokines do not only bind to their specific cell surface receptors but also interact with components of the extracellular matrix. Mainly in Drosophila, several enzymes were identified, that are involved in glycosaminoglycan synthesis. Mutations in these enzymes mostly result in disturbances of several signaling pathways like hedgehog, wingless, FGF or dpp. In most cases it was, due to these pleiotropic effects, not possible to examine the relevance of matrix interactions for single pathways. The aim of this work was to examine the relevance of matrix interactions for the TGF-ß superfamily member DPP. Based on the fact that DPP is highly homologous to human BMP-2, the basic N-terminus of mature DPP was mutated, which has been shown to contain a heparin-binding site in BMP-2. Thus, a wildtype variant (D-MYC), a deletion variant (D-DEL), which lacked the whole basic part of the N-terminus and a duplication variant (D-DUP), which contained a second copy of the basic core moitiv, were generated. In order to characterise the variants biochemically, they were expressed in E.coli and refolded in a bioactive form. In chicken limbbud assay, the deletion variant was much more active than the wildtype variant, comparable to data of BMP-2. By means of biacore mesurements with the immobilised ectodomain of the high affinity type I receptor thick veins, it could be demonstrated, that the variants differ only in matrix binding and not in their receptor affinity. Different matrix binding was shown by Heparin FPLC. The biological relevance of the matrix interaction of DPP was examined in transgenic flies. To allow expression of the different variants under the control of various Gal4 driver lines, they were cloned behind an UAS-promoter site. In early tracheal development, a strong dependence of DPP signaling on matrix binding was observed. While ectopic expression of the deletion variant caused only minor defects, the branching pattern was strongly disturbed by overexpression of wildtype and duplication variant. Ubiquitous expression of the variants in the wing imaginal disc caused overproliferation of the disc and expansion of the omb target gene expression. The extent of phenotypes correlated with the matrix binding ability of the variants. Corresponding disturbances of the wing vein pattern was observed in adult flies. By the crossing of different dpp allels, transheterozygous animals were created, that lack dpp only in imaginal discs. Expression of the variants under the control of a suitable dpp-Gal4 driver line revealed insights into the biological relevance of matrix binding on DPP gradient formation and specific target gene activation in wing imaginal discs. It was shown, that all variants were able to generate a functional DPP gradient with correct expression of the target genes omb and spalt. Again a correlation between extent of target gene domains and matrix binding ability of the corresponding variants was found. Thus by mutating the N-terminus of DPP, it could be shown that this is responsible for DPP`s matrix interaction. Also the relevance of matrix binding of DPP in different tissues was examined. It turned out, that the reorganisation of tracheal branching by DPP strongly depends on matrix interactions wheras the establishing of a gradient in wing imaginal discs depends only gradually on matrix interactions. Based on these data a model for the action of DPP/TGFßs as morphogens was established. While a deletion of matrix binding leads to a decrease in specific bioactivity of the cytokine, the latter is increased by additional matrix binding sites.
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.
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.
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.
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.
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.
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.
In this thesis, synaptic transmission was studied electrophysiologically at an invertebrate model synapse, the neuromuscular junction of the Drosophila 3rd instar wandering larvae. In the first part, synaptic function is characterized at the neuromuscular junction in fly lines which are null mutants for the synaptic proteins “the synapse associated protein of 47 kDa” (Sap-47156), Synapsin (Syn97), the corresponding double mutant (Sap-47156, Syn97), a null mutant for an as yet uncharacterized Drosophila SR protein kinase, the Serine-Arginine protein kinase 3 (SRPK3), and the Löchrig (Loe) mutant which shows a strong neurodegenerative phenotype. Intracellular voltage recordings from larval body wall muscles 6 and 7 were performed to measure amplitude and frequency of spontaneous single vesicle fusion events (miniature excitatory junction potentials or mEJPs). Evoked excitatory junction potentials (eEJPs) at different frequencies and calcium concentrations were also measured to see if synaptic transmission was altered in mutants which lacked these synaptic proteins. In addition, structure and morphology of presynaptic boutons at the larval neuromuscular junction were examined immunohistochemically using monoclonal antibodies against different synaptic vesicle proteins (SAP-47, CSP, and Synapsin) as well as the active zone protein Bruchpilot. Synaptic physiology and morphology was found to be similar in all null mutant lines. However, Löchrig mutants displayed an elongated bouton morphology, a significant shift towards larger events in mEJP amplitude frequency histograms, and increased synaptic facilitation during a 10 Hz tetanus. These deficits suggest that Loe mutants may have a defect in some aspect of synaptic vesicle recycling. The second part of this thesis involved the electrophysiological characterization of heterologously expressed light activated proteins at the Drosophila neuromuscular junction. Channelrhodopsin-2 (ChR2), a light gated ion channel, and a photoactivated adenylate cyclase (PAC) were expressed in larval motor neurons using the UAS-Gal4 system. Single EJPs could be recorded from muscles 15, 16, and 17 when larva expressing ChR2 were illuminated with short (100 ms) light pulses, whereas long light pulses (10 seconds) resulted in trains of EJPs with a frequency of around 25 Hz. Larva expressing PAC in preparations where motor neurons were cut from the ventral ganglion displayed a significant increase in mEJP frequency after a 1 minute exposure to blue light. Evoked responses in low (.2 mM) calcium were also significantly increased when PAC was stimulated with blue light. When motor nerves were left intact, PAC stimulation resulted in light evoked EJPs in muscles 6 and 7 in a manner consistent with RP3 motor neuron activity. ChR2 and PAC are therefore useful and reliable tools for manipulating neuronal activity in vivo.
Past experience contributes to behavioural organization mainly via learning: Animals learn otherwise ordinary cues as predictors for biologically significant events. This thesis studies such predictive, associative learning, using the fruit fly Drosophila melanogaster. I ask two main questions, which complement each other: One deals with the processing of those cues that are to be learned as predictors for an important event; the other one deals with the processing of the important event itself, which is to be predicted. Do fruit flies learn about combinations of olfactory and visual cues? I probe larval as well as adult fruit flies for the learning about combinations of olfactory and visual cues, using a so called ‘biconditional discrimination’ task: During training, one odour is paired with reinforcement only in light, but not in darkness; the other odour in turn is reinforced only in darkness, but not in light. Thus, neither the odours nor the visual conditions alone predict reinforcement, only combinations of both do. I find no evidence that either larval or adult fruit flies were to solve such task, speaking against a cross-talk between olfactory and visual modalities. Previous studies however suggest such cross-talk. To reconcile these results, I suggest classifying different kinds of interaction between sensory modalities, according to their site along the sensory-motor continuum: I consider an interaction ‘truly’ cross-modal, if it is between the specific features of the stimuli. I consider an interaction ’amodal’ if it instead engages the behavioural tendencies or ‘values’ elicited by each stimulus. Such reasoning brings me to conclude that different behavioural tasks require different kinds of interaction between sensory modalities; whether a given kind of interaction will be found depends on the neuronal infrastructure, which is a function of the species and the developmental stage. Predictive learning of pain-relief in fruit flies Fruit flies build two opposing kinds of memory, based on an experience with electric shock: Those odours that precede shock during training are learned as predictors for punishment and are subsequently avoided; those odours that follow shock during training on the other hand are learned as signals for relief and are subsequently approached. I focus on such relief learning. I start with a detailed parametric analysis of relief learning, testing for reproducibility as well as effects of gender, repetition of training, odour identity, odour concentration and shock intensity. I also characterize how relief memories, once formed, decay. In addition, concerning the psychological mechanisms of relief learning, first, I show that relief learning establishes genuinely associative conditioned approach behaviour and second, I report that it is most likely not mediated by context associations. These results enable the following neurobiological analysis of relief learning; further, they will form in the future the basis for a mathematical model; finally, they will guide the researchers aiming at uncovering relief learning in other experimental systems. Next, I embark upon neurogenetic analysis of relief learning. First, I report that fruit flies mutant for the so called white gene build overall more ‘negative’ memories about an experience with electric shock. That is, in the white mutants, learning about the painful onset of shock is enhanced, whereas learning about the relieving offset of shock is diminished. As they are coherently affected, these two kinds of learning should be in a balance. The molecular mechanism of the effect of white on this balance remains unresolved. Finally, as a first step towards a neuronal circuit analysis of relief learning, I compare it to reward learning and punishment learning. I find that relief learning is distinct from both in terms of the requirement for biogenic amine signaling: Reward and punishment are respectively signalled by octopamine and dopamine, for relief learning, either of these seem dispensible. Further, I find no evidence for roles for two other biogenic amines, tyramine and serotonin in relief learning. Based on these findings I give directions for further research.
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.
Neuroanatomical data in fly brain research are mostly available as spatial gene expression patterns of genetically distinct fly strains. The Drosophila standard brain, which was developed in the past to provide a reference coordinate system, can be used to integrate these data. Working with the standard brain requires advanced image processing methods, including visualisation, segmentation and registration. The previously published VIB Protocol addressed the problem of image registration. Unfortunately, its usage was severely limited by the necessity of manually labelling a predefined set of neuropils in the brain images at hand. In this work I present novel tools to facilitate the work with the Drosophila standard brain. These tools are integrated in a well-known open-source image processing framework which can potentially serve as a common platform for image analysis in the neuroanatomical research community: ImageJ. In particular, a hardware-accelerated 3D visualisation framework was developed for ImageJ which extends its limited 3D visualisation capabilities. It is used for the development of a novel semi-automatic segmentation method, which implements automatic surface growing based on user-provided seed points. Template surfaces, incorporated with a modified variant of an active surface model, complement the segmentation. An automatic nonrigid warping algorithm is applied, based on point correspondences established through the extracted surfaces. Finally, I show how the individual steps can be fully automated, and demonstrate its application for the successful registration of fly brain images. The new tools are freely available as ImageJ plugins. I compare the results obtained by the introduced methods with the output of the VIB Protocol and conclude that our methods reduce the required effort five to ten fold. Furthermore, reproducibility and accuracy are enhanced using the proposed tools.
This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.
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.
Understanding of complex interactions and events in a nervous system, leading from the molecular level up to certain behavioural patterns calls for interdisciplinary interactions of various research areas. The goal of the presented work is to achieve such an interdisciplinary approach to study and manipulate animal behaviour and its underlying mechanisms. Optical in vivo imaging is a new constantly evolving method, allowing one to study not only the local but also wide reaching activity in the nervous system. Due to ease of its genetic accessibility Drosophila melanogaster represents an extraordinary experimental organism to utilize not only imaging but also various optogenetic techniques to study the neuronal underpinnings of behaviour. In this study four genetically encoded sensors were used to investigate the temporal dynamics of cAMP concentration changes in the horizontal lobes of the mushroom body, a brain area important for learning and memory, in response to various physiological and pharmacological stimuli. Several transgenic lines with various genomic insertion sites for the sensor constructs Epac1, Epac2, Epac2K390E and HCN2 were screened for the best signal quality, one line was selected for further experiments. The in vivo functionality of the sensor was assessed via pharmacological application of 8-bromo-cAMP as well as Forskolin, a substance stimulating cAMP producing adenylyl cyclases. This was followed by recording of the cAMP dynamics in response to the application of dopamine and octopamine, as well as to the presentation of electric shock, odorants or a simulated olfactory signal, induced by acetylcholine application to the observed brain area. In addition the interaction between the shock and the simulated olfactory signal by simultaneous presentation of both stimuli was studied. Preliminary results are supporting a coincidence detection mechanism at the level of the adenylyl cyclase as postulated by the present model for classical olfactory conditioning. In a second series of experiments an effort was made to selecticvely activate a subset of neurons via the optogenetic tool Channelrhodopsin (ChR2). This was achieved by recording the behaviour of the fly in a walking ball paradigm. A new method was developed to analyse the walking behaviour of the animal whose brain was made optically accessible via a dissection technique, as used for imaging, thus allowing one to target selected brain areas. Using the Gal4-UAS system the protocerebral bridge, a substructure of the central complex, was highlighted by expressing the ChR2 tagged by fluorescent protein EYFP. First behavioural recordings of such specially prepared animals were made. Lastly a new experimental paradigm for single animal conditioning was developed (Shock Box). Its design is based on the established Heat Box paradigm, however in addition to spatial and operant conditioning available in the Heat Box, the design of the new paradigm allows one to set up experiments to study classical and semioperant olfactory conditioning, as well as semioperant place learning and operant no idleness experiments. First experiments involving place learning were successfully performed in the new apparatus.
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.
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