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The learned helplessness phenomenon is a specific animal behavior induced by prior exposure to uncontrollable aversive stimuli. It was first found by Seligman and Maier (1967) in dogs and then has been reported in many other species, e.g. in rats (Vollmayr and Henn, 2001), in goldfishes (Padilla, 1970), in cockroaches (Brown, 1988) and also in fruit flies (Brown, 1996; Bertolucci, 2008). However, the learned helplessness effect in fruit flies (Drosophila melanogaster) has not been studied in detail. Thus, in this doctoral study, we investigated systematically learned helplessness behavior of Drosophila for the first time.
Three groups of flies were tested in heatbox. Control group was in the chambers experiencing constant, mild temperature. Second group, master flies were punished in their chambers by being heated if they stopped walking for 0.9s. The heat pulses ended as soon as they resumed walking again. A third group, the yoked fly, was in their chambers at the same time. However, their behavior didn’t affect anything: yoked flies were heated whenever master flies did, with same timing and durations. After certain amount of heating events, yoked flies associated their own behavior with the uncontrollability of the environment. They suppressed their innate responses such as reducing their walking time and walking speed; making longer escape latencies and less turning around behavior under heat pulses. Even after the conditioning phase, yoked flies showed lower activity level than master and control flies. Interestingly, we have also observed sex dimorphisms in flies. Male flies expressed learned helplessness not like female flies. Differences between master and yoked flies were smaller in male than in female flies. Another interesting finding was that prolonged or even repetition of training phases didn’t enhance learned helplessness effect in flies.
Furthermore, we investigated serotonergic and dopaminergic nervous systems in learned helplessness. Using genetic and pharmacological manipulations, we altered the levels of serotonin and dopamine in flies’ central nervous system. Female flies with reduced serotonin concentration didn’t show helpless behavior, while the learned helplessness effect in male flies seems not to be affected by a reduction of serotonin. Flies with lower dopamine level do not display the learned helplessness effect in the test phase, suggesting that with low dopamine the motivational change in learned helplessness in Drosophila may decline faster than with a normal dopamine level.
Chemical neurotransmission is a complex process of central importance for nervous system function. It is thought to be mediated by the orchestration of hundreds of proteins for its successful execution. Several synaptic proteins have been shown to be relevant for neurotransmission and many of them are highly conserved during evolution- suggesting a universal mechanism for neurotransmission. This process has checkpoints at various places like, neurotransmitter uptake into the vesicles, relocation of the vesicles to the vicinity of calcium channels in order to facilitate Ca2+ induced release thereby modulating the fusion probability, formation of a fusion pore to release the neurotransmitter and finally reuptake of the vesicles by endocytosis. Each of these checkpoints has now become a special area of study and maintains its own importance for the understanding of the overall process. Ca2+ induced release occurs at specialized membrane structures at the synapse known as the active zones. These are highly ordered electron dense grids and are composed of several proteins which assist the synaptic vesicles in relocating in the vicinity of Ca2+ channels thereby increasing their fusion probability and then bringing about the vesicular fusion itself. All the protein modules needed for these processes are thought to be held in tight arrays at the active zones, and the functions of a few have been characterized so far at the vertebrate active zones. Our group is primarily interested in characterizing the molecular architecture of the Drosophila synapse. Due to its powerful genetics and well-established behavioural assays Drosophila is an excellent system to investigate neuronal functioning. Monoclonal antibodies (MABs) from a hybridoma library against Drosophila brain are routinely used to detect novel proteins in the brain in a reverse genetic approach. Upon identification of the protein its encoding genetic locus is characterized and a detailed investigation of its function is initiated. This approach has been particularly useful to detect synaptic proteins, which may go undetected in a forward genetic approach due to lack of an observable phenotype. Proteins like CSP, Synapsin and Sap47 have been identified and characterized using this approach so far. MAB nc82 has been one of the shortlisted antibodies from the same library and is widely used as a general neuropil marker due to the relative transparency of immunohistochemical whole mount staining obtained with this antibody. A careful observation of double stainings at the larval neuromuscular junctions with MAB nc82 and other pre and post-synaptic markers strongly suggested an active zone localization of the nc82 antigen. Synaptic architecture is well characterized in Drosophila at the ultrastructural level. However, molecular details for many synaptic components and especially for the active zone are almost entirely unknown. A possible localization at the active zone for the nc82 antigen served as the motivation to initiate its biochemical characterization and the identification of the encoding gene. In the present thesis it is shown by 2-D gel analysis and mass spectrometry that the nc82 antigen is a novel active zone protein encoded by a complex genetic locus on chromosome 2R. By RT-PCR exons from three open reading frames previously annotated as separate genes are demonstrated to give rise to a transcript of at least 5.5 kb. Northern blots produce a prominent signal of 11 kb and a weak signal of 2 kb. The protein encoded by the 5.5 kb transcript is highly conserved amongst insects and has at its N-terminus significant homology to the previously described vertebrate active zone protein ELKS/ERC/CAST. Bioinformatic analysis predicts coiled-coil domains spread all over the sequence and strongly suggest a function involved in organizing or maintaining the structure of the active zone. The large C-terminal region is highly conserved amongst the insects but has no clear homologues in veretebrates. For a functional analysis of this protein transgenic flies expressing RNAi constructs under the control of the Gal4 regulated enhancer UAS were kindly provided by the collaborating group of S.Sigrist (Gِttingen). A strong pan-neuronal knockdown of the nc82 antigen by transgenic RNAi expression leads to embryonic lethality. A relatively weaker RNAi expression results in behavioural deficits in adult flies including unstable flight and impaired walking behavior. Due to this peculiar phenotype as observed in the first knockdown studies the gene was named “bruchpilot” (brp) encoding the protein “Bruchpilot (BRP)” (German for crash pilot). A pan-neuronal as well as retina specific downregulation of this protein results in loss of ON and OFF transients in ERG recordings indicating dysfunctional synapses. Retina specific downregulation also shows severely impaired optomotor behaviour. Finally, at an ultrastructural level BRP downregulation seems to impair the formation of the characteristic T-shaped synaptic ribbons at the active zones without significantly altering the overall synaptic architecture (in collaboration with E.Asan). Vertebrate active zone protein Bassoon is known to be involved in attaching the synaptic ribbons to the active zones as an adapter between active zone proteins RIBEYE and ERC/CAST. A mutation in Bassoon results in a floating synaptic ribbon phenotype. No protein homologous to Bassoon has been observed in Drosophila. BRP downregulation also results in absence of attached synaptic ribbons at the active zones. This invites the speculation of an adapter like function for BRP in Drosophila. However, while Bassoon mutant mice are viable, BRP deficit in addition to the structural phenotype also results in severe behavioural and physiological anomalies and even stronger downregulation causes embryonic lethality. This therefore suggests an additional and even more important role for BRP in development and normal functioning of synapses in Drosophila and also in other insects. However, how BRP regulates synaptic transmission and which other proteins are involved in this BRP dependant pathway remains to be investigated. Such studies certainly will attract prominent attention in the future.
ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1
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.
Structural and functional modifications of synaptic connections (“synaptic plasticity”) are believed to mediate learning and memory processes. Thus, molecular mechanisms of how synapses assemble in both structural and functional terms are relevant for our understanding of neuronal development as well as the processes of learning and memory. Synapses form by an asymmetric association of highly specialized membrane domains: at the presynaptic active zone transmitter filled vesicles fuse, while transmitter receptors at the opposite postsynaptic density sense this signal. By genetic analysis, matrix proteins of active zones from various families have been shown to be important for fast vesicle fusion, and were suggested to contribute to synapse stability and assembly. The Sigrist lab in collaboration with the Buchner lab previously had shown that the large scaffold protein Bruchpilot (Brp) is essential for both the structural and functional integrity of active zones and for synaptic plasticity in Drosophila melanogaster. The work described in this thesis investigated several candidate proteins which appear to be involved in preand postsynaptic function, as summarized in the following: (1) DREP-2 (DEF45 related protein-2) had been found by co-immunoprecipitations with anti-Brp antibodies by Dr. Manuela Schmidt (unpublished data). Mutants and antibodies for the further study of DREP- 2 were generated in this thesis. Yeast two hybrid results suggest that DREP-2 might interact with dynein light chain 2, while in vivo imaging indicates that DREP-2 might be involved in bidirectional axonal transport. (2) Coimmunoprecipitation and pull down experiments suggested that the ARFGAP [ADP-ribosylation factor (ARF)-directed GTPase activating protein (GAP)] protein GIT (G-protein coupled receptor kinase interacting protein) could interact with the endocytosis associated molecule Stoned B (StnB). Mutants in the dgit gene showed an accumulation of large size vesicles, membrane intermediates and decreased vesicle density at the 3rd instar larval neuromuscular junction (NMJ) by electron microscopy (EM). The phenotypes accumulation of large size vesicles and membrane intermediates could be rescued partially by expression of Drosophila GIT (DGIT) or human GIT in dgit mutant background. Furthermore, by immunofluorescence the dgit mutant shows specifically decreased levels of StnB, which could be restored partially by the expression of DGIT. These results strongly support the suggestion that DGIT interacts with StnB, which is involved in the regulation of vesicle size, endocytosis or recycling of synaptic vesicles (SVs). Furthermore, the dgit mutants also showed signs of a mislocalization of the presynaptic protein Brp relative to the postsynaptic protein GluRIID, which could be rescued by expression of DGIT or human GIT in the dgit mutant background, but not by StnB. These results suggest that GIT on one hand executes roles in the regulation of synaptic vesicle endocytosis, but potentially also has structural roles for synapse assembly (3) Djm-1 is a candidate locus to mediate mental retardation in human patients when it is mutated. As a first step towards an understanding of the mechanistic role of DJM-1, Drosophila genetics were used to address DJM-1 function. So far, however, the djm-1 mutant generated in this thesis did not show a nervous system phenotype.
Sugar reward learning in Drosophila : neuronal circuits in Drosophila associative olfactory learning
(2006)
Genetic intervention in the fly Drosophila melanogaster has provided strong evidence that the mushroom bodies of the insect brain act as the seat of memory traces for aversive and appetitive olfactory learning (reviewed in Heisenberg, 2003). In flies, electroshock is mainly used as negative reinforcer. Unfortunately this fact complicates a comparative consideration with other inscets as most studies use sugar as positive reinforcer. For example, several lines of evidence from honeybee and moth have suggested another site, the antennal lobe, to house neuronal plasticity underlying appetitive olfactory memory (reviewed in Menzel, 2001; Daly et al., 2004). Because of this I focused my work mainly on appetitive olfactory learning. In the first part of my thesis, I used a novel genetic tool, the TARGET system (McGuire et al., 2003), which allows the temporally controlled expression of a given effector gene in a defined set of cells. Comparing effector genes which either block neurotransmission or ablate cells showed important differences, revealing that selection of the appropriate effector gene is critical for evaluating the function of neural circuits. In the second part, a new engram of olfactory memory in the Drosophila projection neurons is described by restoring Rutabaga adenlylate cyclase (rut-AC) activity specifically in these cells. Expression of wild-type rutabaga in the projection neurons fully rescued the defect in sugar reward memory, but not in aversive electric shock memory. No difference was found in the stability of the appetitive memories rescued either in projection neurons or Kenyon cells. In the third part of the thesis I tried to understand how the reinforcing signals for sugar reward are internally represented. In the bee Hammer (1993) described a single octopaminergic neuron – called VUMmx1 – that mediates the sugar stimulus in associative olfactory reward learning. Analysis of single VUM neurons in the fly (Selcho, 2006) identified a neuron with a similar morphology as the VUMmx1 neuron. As there is a mutant in Drosophila lacking the last enzymatic step in octopamine synthesis (Monastirioti et al., 1996), Tyramine beta Hydroxylase, I was able to show that local Tyramine beta Hydroxylase expression successfully rescued sugar reward learning. This allows to conclude that about 250 cells including the VUM cluster are sufficient for mediating the sugar reinforcement signal in the fly. The description of a VUMmx1 similar neuron and the involvement of the VUM cluster in mediating the octopaminergic sugar stimulus are the first steps in establishing a neuronal map for US processing in Drosophila. Based on this work several experiments are contrivable to reach this ultimate goal in the fly. Taken together, the described similiarities between Drosophila and honeybee regarding the memory organisation in MBs and PNs and the proposed internal representation of the sugar reward suggest an evolutionarily conserved mechanism for appetitive olfactory learning in insects.
This study explores novelty choice, a behavioral paradigm for the investigation of visual pattern recognition and learning of the fly Drosophila melanogaster in the flight simulator. Pattern recognition in novelty choice differs significantly from pattern recognition studied by heat conditioning, although both paradigms use the same test. Out of the four pattern parameters that the flies can learn in heat conditioning, novelty choice can be shown for height (horizontal bars differing in height), size and vertical compactness but not for oblique bars oriented at +/- 45°. Upright and inverted Ts [differing in their centers of gravity (CsOG) by 13°] that have been extensively used for heat conditioning experiments, do not elicit novelty choice. In contrast, horizontal bars differing in their CsOG by 13° do elicit novelty choice; so do the Ts after increasing their CsOG difference from 13° to 23°. This indicates that in the Ts the heights of the CsOG are not the only pattern parameters that matter for the novelty choice behavior. The novelty choice and heat conditioning paradigms are further differentiated using the gene rutabaga (rut) coding for a type 1 adenylyl cyclase. This protein had been shown to be involved in memory formation in the heat conditioning paradigm. Novelty choice is not affected by mutations in the rut gene. This is in line with the finding that dopamine, which in olfactory learning is known to regulate Rutabaga via the dopamine receptor Dumb in the mushroom bodies, is dispensable for novelty choice. It is concluded that in novelty choice the Rut cAMP pathway is not involved. Novelty choice requires short term working memory, as has been described in spatial orientation during locomotion. The protein S6KII that has been shown to be involved in visual orientation memory in walking flies is found here to be also required for novelty choice. As in heat conditioning the central complex plays a major role in novelty choice. The S6KII mutant phenotype for height can be rescued in some subsets of the ring neurons of the ellipsoid body. In addition the finding that the ellipsoid body mutants ebo678 and eboKS263 also show a mutant phenotype for height confirm the importance of ellipsoid body for height novelty choice. Interestingly some neurons in the F1 layer of the fan-shaped body are necessary for height novelty choice. Furthermore, different novelty choice phenotypes for different pattern parameters are found with and without mushroom bodies. Mushroom bodies are required in novelty choice for size but they are dispensable for height and vertical compactness. This special circuit requirement for the size parameter in novelty choice is found using various means of interference with mushroom body function during development or adulthood.
Zars and co-workers were able to localize an engram of aversive olfactory memory to the mushroom bodies of Drosophila (Zars et al., 2000). In this thesis, I followed up on this finding in two ways. Inspired by Zars et al. (2000), I first focused on the whether it would also be possible to localize memory extinction.While memory extinction is well established behaviorally, little is known about the underlying circuitry and molecular mechanisms. In extension to the findings by Zars et al (2000), I show that aversive olfactory memories remain localized to a subset of mushroom body Kenyon cells for up to 3 hours. Extinction localizes to the same set of Kenyon cells. This common localization suggests a model in which unreinforced presentations of a previously learned odorant intracellularly antagonizes the signaling cascades underlying memory formation. The second part also targets memory localization, but addresses appetitive memory. I show that memories for the same olfactory cue can be established through either sugar or electric shock reinforcement. Importantly, these memories localize to the same set of neurons within the mushroom body. Thus, the question becomes apparent how the same signal can be associated with different events. It is shown that two different monoamines are specificaly necessary for formation of either of these memories, dopamine in case of electric shock and octopamine in case of sugar memory, respectively. Taking the representation of the olfactory cue within the mushroom bodies into account, the data suggest that the two memory traces are located in the same Kenyon cells, but in separate subcellular domains, one modulated by dopamine, the other by octopamine. Taken together, this study takes two further steps in the search for the engram. (1) The result that in Drosophila olfactory learning several memories are organized within the same set of Kenyon cells is in contrast to the pessimism expressed by Lashley that is might not be possible to localize an engram. (2) Beyond localization, a possibible mechanism how several engrams about the same stimulus can be localized within the same neurons might be suggested by the models of subcellular organisation, as postulated in case of appetitive and aversive memory on the one hand and acquisition and extinction of aversive memory on the other hand.
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.
All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism.
The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons.
To further study the functional hierarchy within the clock network and to monitor the “ticking clock“ over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.
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.
The change of day and night is one of the challenges all organisms are exposed to, as they have to adjust their physiology and behavior in an appropriate way. Therefore so called circadian clocks have evolved, which allow the organism to predict these cyclic changes of day and night. The underlying molecular mechanism is oscillating with its endogenous period of approximately 24 hours in constant conditions, but as soon as external stimuli, so called Zeitgebers, are present, the clocks adjust their period to exactly 24h, which is called entrainment. Studies in several species, including humans, animals and plants, showed that light is the most important Zeitgeber synchronizing physiology and behavior to the changes of day and night. Nevertheless also other stimuli, like changes in temperature, humidity or social interactions, are powerful Zeitgebers for entraining the clock. This thesis will focus on the question, how light influences the locomotor behavior of the fly in general, including a particular interest on the entrainment of the circadian clock. As a model organism Drosophila melanogaster was used.
During the last years several research groups investigated the effect of light on the circadian clock and their results showed that several light input pathways to the clock contribute to wild-type behavior. Most of the studies focused on the photopigment Cryptochrome (CRY) which is expressed in about half of the 150 clock neurons in the fly. CRY is activated by light, degrades the clock protein Timeless (TIM) and hence entrains the clock to the light-dark (LD)-cycle resulting from changes of day and night. However, also flies lacking CRY are still able to entrain their clock mechanism as well as their activity-rest-rhythm to LD-cycles, clearly showing that the visual system of the fly also contributes to clock synchronization. The mechanism how light information from the visual system is transferred to the clock is so far still unknown. This is also true for so-called masking-effects which are changes in the behavior of the animal that are directly initiated by external stimuli and therefore independent of the circadian clock. These effects complement the behavior of the animals as they enable the fly to react quickly to changes in the environment even during the clock-controlled rest state.
Both of these behavioral features were analyzed in more detail in this study. On the one hand, we investigated the influence of the compound eyes on the entrainment of the clock neurons and on the other hand, we tried to separate clock-controlled behavior from masking. To do so "nature-like" light conditions were simulated allowing the investigation of masking and entrainment within one experiment. The simulation of moonlight and twilight conditions caused significant changes in the locomotor behavior. Moonlit nights increased nocturnal activity levels and shifted the morning (M) and evening (E) activity bouts into the night. The opposite was true for the investigation of twilight, as the activity bouts were shifted into the day. The simulation of twilight and moonlight within the same experiment further showed that twilight appears to dominate over moonlight, which is in accordance to the assumption that twilight in nature is one of the key signals to synchronize the clock as the light intensity during early dawn rises similarly in every season. By investigating different mutants with impaired visual system we showed that the compound eyes are essential for the observed behavioral adaptations. The inner receptor cells (R7 and R8) are important for synchronizing the endogenous clock mechanism to the changes of day and night. In terms of masking, a complex interaction of all receptor cells seems to adjust the behavioral pattern, as only flies lacking photopigments in inner and outer receptor cells lacked all masking effects. However, not only the compound eyes seem to contribute to rhythmic activity in moonlit nights. CRY-mutant flies shift their E activity bout even more into the night than wild-type flies do. By applying Drosophila genetics we were able to narrow down this effect to only four CRY expressing clock neurons per hemisphere. This implies that the compound eyes and CRY in the clock neurons have antagonistic effects on the timing of the E activity bout. CRY advances activity into the day, whereas the compound eyes delay it. Therefore, wild-type behavior combines both effects and the two light inputs might enable the fly to time its activity to the appropriate time of day.
But CRY expression is not restricted to the clock neurons as a previous study showed a rather broad distribution within the compound eyes. In order to investigate its function in the eyes we collaborated with Prof. Rodolfo Costa (University of Padova). In our first study we were able to show that CRY interacts with the phototransduction cascade and thereby influences visual behavior like phototaxis and optomotor response. Our second study showed that CRY in the eyes affects locomotor activity rhythms. It appears to contribute to light sensation without being a photopigment per se. Our results rather indicate that CRY keeps the components of the phototransduction cascade close to the cytoskeleton, as we identified a CRY-Actin interaction in vitro. It might therefore facilitate the transformation of light energy into electric signals.
In a further collaboration with Prof. Orie Shafer (University of Michigan) we were able to shed light on the significance of the extraretinal Hofbauer-Buchner eyelet for clock synchronization. Excitation of the eyelet leads to Ca2+ and cAMP increases in specific clock neurons, consequently resulting in a shift of the flies´ rhythmic activity.
Taken together, the experiments conducted in this thesis revealed new functions of different eye structures and CRY for fly behavior. We were furthermore able to show that masking complements the rhythmic behavior of the fly, which might help to adapt to natural conditions.
Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The ‘decision’ whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food – being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment – being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the ‘decision’ process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.
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.
According to a changing environment it is crucial for animals to make experience and learn about it. Sensing, integrating and learning to associate different kinds of modalities enables animals to expect future events and to adjust behavior in the way, expected as the most profitable. Complex processes as memory formation and storage make it necessary to investigate learning and memory on different levels. In this context Drosophila melanogaster represents a powerful model organism. As the adult brain of the fly is still quite complex, I chose the third instar larva as model - the more simple the system, the easier to isolate single, fundamental principles of learning. In this thesis I addressed several kinds of questions on different mechanism of olfactory associative and synaptic plasiticity in Drosophila larvae. I focused on short-term memory throughout my thesis. First, investigating larval learning on behavioral level, I developed a one-odor paradigm for olfactory associative conditioning. This enables to estimate the learnability of single odors, reduces the complexity of the task and simplify analyses of "learning mutants". It further allows to balance learnability of odors for generalization-type experiments to describe the olfactory "coding space". Furthermore I could show that innate attractiveness and learnability can be dissociated and found finally that paired presentation of a given odor with reward increase performance, whereas unpaired presentations of these two stimuli decrease performance, indicating that larva are able to learn about the presence as well as about the absence of a reward. Second, on behavioral level, together with Thomas Niewalda and colleagues we focussed on salt processing in the context of choice, feeding and learning. Salt is required in several physiological processes, but can neither be synthesized nor stored. Various salt concentrations shift the valence from attraction to repulsion in reflexive behaviour. Interestingly, the reinforcing effect of salt in learning is shifted by more than one order of magnitude toward higher concentrations. Thus, the input pathways for gustatory behavior appear to be more sensitive than the ones supporting gustatory reinforcement, which is may be due to the dissociation of the reflexive and the reinforcing signalling pathways of salt. Third, in cooperation with Michael Schleyer we performed a series of behavioral gustatory, olfactory preference tests and larval learning experiments. Based on the available neuroanatomical and behavioral data we propose a model regarding chemosensory processing, odor-tastant memory trace formation and the 'decision' like process. It incorporates putative sites of interaction between olfactory and gustatory pathways during the establishment as well as behavioral expression of odor-tastant memory. We claim that innate olfactory behavior is responsive in nature and suggest that associative conditioned behavior is not a simple substitution like process, but driven more likely by the expectation of its outcome. Fourth, together with Birgit Michels and colleagues we investigated the cellular site and molecular mode of Synapsin, an evolutionarily conserved, presynaptic vesicular phosphoprotein and its action in larval learning. We confirmed a previously described learning impairment upon loss of Synapsin. We localized this Synapsin dependent memory trace in the mushroom bodies, a third-order "cortical" brain region, and could further show on molecular level, that Synapsin is as a downstream element of the AC-cAMP-PKA signalling cascade. This study provides a comprehensive chain of explanation from the molecular level to an associative behavioral change. Fifth, in the main part of my thesis I focused on molecular level on another synaptic protein, the Synapse associated protein of 47kDa (Sap47) and its role in larval behavior. As a member of a phylogenetically conserved gene family of hitherto unknown function. It is localized throughout the whole neuropil of larval brains and associated with presynaptic vesicles. Upon loss of Sap47 larvae exhibit normal sensory detection of the to-be-associated stimuli as well as normal motor performance and basic synaptic transmission. Interestingly, short-term plasticity is distorted and odorant–tastant associative learning ability is reduced. This defect in associative function could be rescued by restoring Sap47 expression. Therefore, this report is the first to suggest a function for Sap47 and specifically argues that Sap47 is required for synaptic as well as for behavioral plasticity in Drosophila larva. This prompts the question whether its homologs are required for synaptic and behavioral plasticity also in other species. Further in the last part of my thesis I contributed to the study of Ayse Yarali. Her central topic was the role of the White protein in punishment and relief learning in adult flies. Whereas stimuli that precede shock during training are subsequently avoided as predictors for punishment, stimuli that follow shock during training are later on approached, as they predict relief. Concerning the loss of White we report that pain-relief learning as well as punishment learning is changed. My contribution was a comparison between wild type and the white1118 mutant larvae in odor-reward learning. It turned out that a loss of White has no effect on larval odorant-tastant learning. This study, regarding painrelief learning provides the very first hints concerning the genetic determinants of this form of learning.
There is such vast amount of visual information in our surroundings at any time that filtering out the important information for further processing is a basic requirement for any visual system. This is accomplished by deploying attention to focus on one source of sensory inputs to the exclusion of others (Luck and Mangun 2009). Attention has been studied extensively in humans and non human primates (NHPs). In Drosophila, visual attention was first demonstrated in 1980 (Wolf and Heisenberg 1980) but this field remained largely unexplored until recently. Lately, however, studies have emerged that hypothesize the role of attention in several behaviors but do not specify the characteristic properties of attention. So, the aim of this research was to characterize the phenomenon of visual attention in wild-type Drosophila, including both externally cued and covert attention using tethered flight at a torque meter. Development of systematic quantifiable behavioral tests was a key aspect for this which was not only important for analyzing the behavior of a population of wild-type flies but also for comparing the wild-type flies with mutant flies. The latter would help understand the molecular, genetic, and neuronal bases of attention. Since Drosophila provides handy genetic tools, a model of attention in Drosophila will serve to the greater questions about the neuronal circuitry and mechanisms involved which might be analogous to those in primates. Such a model might later be used in research involving disorders of attention. Attention can be guided to a certain location in the visual field by the use of external cues. Here, using visual cues the attention of the fly was directed to one or the other of the two visual half-fields. A simple yet robust paradigm was designed with which the results were easily quantifiable. This paradigm helped discover several interesting properties of the cued attention, the most substantial one being that this kind of external guidance of attention is restricted to the lower part of the fly’s visual field. The guiding cue had an after-effect, i.e. it could occur at least up to 2 seconds before the test and still bias it. The cue could also be spatially separated from the test by at least 20° and yet attract the attention although the extent of the focus of attention (FoA) was smaller than one lower visual half-field. These observations excluded the possibility of any kind of interference between the test and the cue stimuli. Another interesting observation was the essentiality of continuous visibility of the test stimulus but not the cue for effective cuing. When the contrast of the visual scene was inverted, differences in response frequencies and cuing effects were observed. Syndirectional yaw torque responses became more frequent than the antidirectional responses and cuing was no longer effective in the lower visual field with inverted contrast. Interestingly, the test stimulus with simultaneous displacement of two stripes not only effectuated a phasic yaw torque response but also a landing response. A 50 landing response was produced in more than half of the cases whenever a yaw torque response was produced. Elucidation of the neuronal correlates of the cued attention was commenced. Pilot experiments with hydroxyurea (HU) treated flies showed that mushroom bodies were not required for the kind of guidance of attention tested in this study. Dopamine mutants were also tested for the guidance of attention in the lower visual field. Surprisingly, TH-Gal4/UAS-shits1 flies flew like wild-type flies and also showed normal optomotor response during the initial calibration phase of the experiment but did not show any phasic yaw torque or landing response at 18 °C, 25 °C or 30 °C. dumb2 flies that have almost no D1 dopamine receptor dDA1 expression in the mushroom bodies and the central complex (Kim et al. 2007) were also tested and like THGal4/ UAS-shits1 flies did not show any phasic yaw torque or landing response. Since the dopamine mutants did not show the basic yaw torque response for the test the role of dopamine in attention could not be deduced. A different paradigm would be needed to test these mutants. Not only can attention be guided through external cues, it can also be shifted endogenously (covert attention). Experiments with the windows having oscillating stripes nicely demonstrated the phenomenon of covert attention due to the production of a characteristic yaw torque pattern by the flies. However, the results were not easily quantifiable and reproducible thereby calling for a more systematic approach. Experiments with simultaneous opposing displacements of two stripes provide a promising avenue as the results from these experiments showed that the flies had a higher tendency to deliver one type of response than when the responses would be produced stochastically suggesting that attention increased this tendency. Further experiments and analysis of such experiments could shed more light on the mechanisms of covert attention in flies.
Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH).
Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions.
Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions.
To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain.
To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies.
To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the Würzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed.
Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown.
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.
Vertebrate and invertebrate visual systems exhibit similarities in early stages of visual processing. For instance, in the human brain, the modalities of color, form and motion are separately processed in parallel neuronal pathways. This basic property is also found in the fly Drosophila melanogaster which has a similar division in color- sensitive and (color blind) motion-sensitive pathways that are determined by two distinct subsets of photoreceptors (the R1-6 and the R7/8 system, respectively). Flies have a highly organized visual system that is characterized by its repetitive, retinotopic organization of four neuropils: the lamina, the medulla, the lobula and the lobula plate. Each of these consists of columns which contain the same set of neurons. In the lamina, axon bundles of six photoreceptors R1-6 that are directed towards the same point in space form columnar structures called cartridges. These are the visual sampling units and are associated with four types of first-order interneuron that receive common input from R1-6: L1, L2, L3 and the amacrine cells (amc, together with their postsynaptic partner T1). They constitute parallel pathways that have been studied in detail at the anatomical level. Little is known, however, about their functional role in processing behaviorally relevant information, e.g. for gaze stabilization, visual course control or the fixation of objects. The availability of a variety of neurogenetic tools for structure-function analysis in Drosophila allowed first steps into the genetic dissection of the neuronal circuitry mediating motion and position detection. In this respect, the choice of the effector turned out to be crucial. Surprisingly, it was found that the clostridial tetanus neurotoxin failed to block mature Drosophila photoreceptor synapses, but caused irreversible damage when expressed during their development. Therefore, the dominant-negative shibire allele shits1 which turned out to be better suited was used for blocking lamina interneurons and thereby analyzing the necessity of the respective pathways. To determine whether the latter were also sufficient for the same behavioral task, the inverse strategy was developed, based on the fact that lamina interneurons express histamine receptors encoded by the ort gene. The specific rescue of ort function in defined channels in an otherwise mutant background allowed studying their sufficiency in a given task. Combining these neurogenetic methods with the optomotor response and object induced orientation behavior as behavioral measures, the aim of the present thesis was to answer the following questions: (a) Which pathways feed into elementary motion detectors and which ones are necessary and/or sufficient for the detection of directional motion? (b) Do pathways exist which specifically mediate responses to unidirectional motion? (c) Which pathways are necessary and/or sufficient for object induced orientation behavior? Some basic properties of the visual circuitry were revealed: The two central cartridge pathways, represented by the large monopolar cells L1 and L2, are key players in motion detection. Under a broad range of stimulatory conditions, the two subsystems are redundant and are able to process motion independently of each other. To detect an impairment when only one of the pathways is intact, one has to drive the system to its operational limits. At low signal to noise ratios, i.e. at low pattern contrast or low background illumination, the L2 pathway has a higher sensitivity. At intermediate pattern contrast, both pathways are specialized in mediating responses to unidirectional motion of opposite stimulus direction. In contrast, neither the L3, nor the amc/T1 pathway is necessary or sufficient for motion detection. While the former may provide position information for orientation, the latter has a modulatory role at intermediate pattern contrast. Orientation behavior turned out to be even more robust than motion vision and may utilize a less sophisticated mechanism, as it does not require a nonlinear comparison of signals from neighboring visual sampling units. The position of objects is processed in several redundant pathways, involving both receptor subsystems. The fixation of objects does not generally require motion vision. However, motion detection improves the fixation of landmarks, especially when these are narrow or have a reduced contrast.
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 this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5’ UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.
PART I Animals need to constantly evaluate their external environment in order to survive. In some cases the internal state of the animal changes to cope with it’s surrounding. In our study we wanted to investigate the role of amines in modulating internal states of Drosophila. We have designed a behavioral paradigm where the flies are fixed in space but can walk on a small styrofoam ball suspended by a gentle stream of air. The walking activity of flies was used as behavioral readout. PART I Animals need to constantly evaluate their external environment in order to survive. In some cases the internal state of the animal changes to cope with it’s surrounding. In our study we wanted to investigate the role of amines in modulating internal states of Drosophila. We have designed a behavioral paradigm where the flies are fixed in space but can walk on a small styrofoam ball suspended by a gentle stream of air. The walking activity of flies was used as behavioral readout. An operant training paradigm was established by coupling one of the walking directions to incidence of heat punishment. We observed that animals quickly realized the contingency of punishment with walking direction and avoided walking in the punished direction in the presence of punishment, but did not continue walking in the unpunished direction in the absence of the punishment. This would indicate that the flies do not form a memory for the punished direction or rapidly erase it under new conditions. On having established the paradigm with heat punishment we have attempted to activate selected subsets of neuronal populations of Drosophila while they were walking on the ball. The selective activation of neurons was achieved by expressing the light-activated ion channel channelrhodopsin-2 (ChR2) using the Gal4-UAS system and coupling the unidirectional walking of the animals on the ball with the incidence of blue light required to activate the channels and depolarize the neurons. The feasibility of this approach was tested by light-activating sugar sensitive gustatory receptor neurons expressing ChR2, we found that when the light was actuated the flies preferred to turn in one direction the optically “rewarded” direction. Next we similarly activated different subsets of aminergic neurons. We observed that in our setup animals avoided to turn in the direction which was coupled to activation of dopaminergic neurons indicating that release of dopamine is disliked by the animals. This is in accordance with associative learning experiments where dopamine is believed to underlie the formation of an association between a neutral conditioned stimulus with the aversive unconditioned stimulus. However, when we activated tyraminergic/octopaminergic neurons we did not observe any directional preference. The activation of dopaminergic and tyraminergic/octopaminergic neurons led to arousal of the animals indicating that we were indeed successful in activating those neurons. Also, the activation of serotonergic neurons did not have any effect on directional preference of the animals. With this newly established paradigm it will be interesting to find out if in insects like in mammals a reward mediating system exists and to test subsets of aminergic or peptidergic neurons that could possibly be involved in a reward signaling system which has not been detected in our study. Also, it would be interesting to localize neuropile regions that would be involved in mediating choice behavior in our paradigm. PART II In collaboration with S. Kneitz (IZKF Wuerzburg) and T. Nuwal we performed genome-wide expression analysis of two pre-synaptic mutants - Synapsin (Syn97) and Synapse associated protein of 47 kDa (Sap47156). The rationale behind these experiments was to identify genes that were up- or down-regulated due to these mutations. The microarray experiments provided us with several candidate genes some of which we have verified by qPCR. From our qPCR analysis we can conclude that out of the verified genes only Cirl transcripts seem to be reproducibly down regulated in Synapsin mutants. The Cirl gene codes for a calcium independent receptor for latrotoxin. Further qPCR experiments need to be performed to verify other candidate genes. The molecular interactions between CIRL and SYN or their genes should now be investigated in detail.
All animals learn in order to cope with challenges imposed on them by their environment. This is true also for both larval and adult fruit flies as exemplified in pavlovian conditioning. The focus of this Thesis is on various aspects of the fruit flies learning ability. My main project deals with two types of learning which we call punishment-learning and pain-relief learning. Punishment learning happens when fruit flies are exposed to an odour which is followed by electric shock. After such training, flies have learned that that odour signals pain and consequently will avoid it in the future. If the sequence of the two stimuli is reversed such that odour follows shock, flies learn the odour as a signal for relief and will later on approach it. I first report a series of experiments investigating qualitative and parametric features of relief-learning; I find that (i) relief learning does result from true associative conditioning, (ii) it requires a relatively high number of training trials, (iii) context-shock training is ineffective for subsequent shock-odour learning. A further question is whether punishment-learning and pain-relief learning share genetic determinants. In terms of genetics, I test a synapsin mutant strain, which lacks all Synapsin protein, in punishment and relief-learning. Punishment learning is significantly reduced, and relief-learning is abolished. Pan-neuronal RNAi-mediated knock-down of Synapsin results in mutant-like phenotypes, confirming the attribution of the phenotype to lack of Synapsin. Also, a rescue of Synapsin in the mushroom body of syn97 mutants restores both punishment- and relief-learning fully, suggesting the sufficiency of Synapsin in the mushroom body for both these kinds of learning. I also elucidate the relationship between perception and physiology in adult fruit flies. I use odour-shock conditioning experiments to identify degrees of similarity between odours; I find that those similarity measures are consistent across generalization and discrimination tasks of diverse difficulty. Then, as collaborator of T. Völler and A. Fiala, I investigate how such behavioural similarity/dissimilarity is reflected at the physiological level. I combine the behaviour data with calcium imaging data obtained by measuring the activity patterns of those odours in either the sensory neurons or the projection neurons at the antennal lobe. Our interpretation of the results is that the odours perceptual similarity is organized by antennal lobe interneurons. In another project I investigate the effect of gustatory stimuli on reflexive behaviour as well as their role as reinforcer in larval learning. Drosophila larvae greatly alter their behaviour in presence of sodium chloride. Increasing salt concentration modulates choice behaviour from weakly appetitive to strongly aversive. A similar concentration-behaviour function is also found for feeding: larval feeding is slightly enhanced in presence of low salt concentrations, and strongly decreased in the presence of high salt concentrations. Regarding learning, relatively weak salt concentrations function as appetitive reinforcer, whereas high salt concentrations function as aversive reinforcer. Interestingly, the behaviour-concentration curves are shifted towards higher concentrations from reflexive behaviour (choice behaviour, feeding) as compared to associative learning. This dissociation may reflect a different sensitivity in the respective sensory-motor circuitry.
Towards localizing the Synapsin-dependent olfactory memory trace in the brain of larval Drosophila
(2008)
Animals need to adapt and modify their behaviour according to a changing environment. In particular, the ability to learn about rewarding or punishing events is crucial for survival. One key process that underlies such learning are modifications of the synaptic connection between nerve cells. This Thesis is concerned with the genetic determinants of such plasticity, and with the site of these modifications along the sensory-to-motor loops in Drosophila olfactory learning. I contributed to the development and detailed parametric description of an olfactory associative learning paradigm in larval fruit flies (Chapter I.1.). The robustness of this learning assay, together with a set of transgenic Drosophila strains established during this Thesis, enabled me to study the role for Synapsin, a presynaptic phosphoprotein likely involved in synaptic plasticity, in this form of learning (Chapter I.2.), and to investigate the cellular site of the corresponding Synapsin-dependent memory trace (Chapter I.3.). These data provide the first comprehensive account to-date of the neurogenetic bases of learning in larval Drosophila. The role for Synapsin was also analyzed with regard to pain-relief learning in adult fruit flies (Chapter II.1.); that is, if an odour precedes an electric shock during training, flies subsequently avoid that odour (‘punishment learning’), whereas presentation of the odour upon the cessation of shock subsequently leads to approach towards the odour (‘relief larning’). Such pain-relief learning was also the central topic of a study concerning the white gene (Chapter II.2.), which as we report does affect pain-relief as well as punishment learning in adult flies, but leaves larval odour-food learning unaffected. These studies regarding pain-relief learning provide the very first hints, in any experimental system, concerning the genetic determinants of this form of learning.
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.
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.
Synaptic plasticity determines the development of functional neural circuits. It is widely accepted as the mechanism behind learning and memory. Among different forms of synaptic plasticity, Hebbian plasticity describes an activity-induced change in synaptic strength, caused by correlated pre- and postsynaptic activity. Additionally, Hebbian plasticity is characterised by input specificity, which means it takes place only at synapses, which participate in activity. Because of its correlative nature, Hebbian plasticity suggests itself as a mechanism behind associative learning.
Although it is commonly assumed that synaptic plasticity is closely linked to synaptic activity during development, the mechanistic understanding of this coupling is far from complete.
In the present study channelrhodopsin-2 was used to evoke activity in vivo, at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation led to increased incorporation of GluR-IIA-type glutamate receptors into postsynaptic receptor fields, thus boosting postsynaptic sensitivity. This phenomenon is input-specific.
Conversely, GluR-IIA was rapidly removed from synapses at which neurotransmitter release failed to evoke substantial postsynaptic depolarisation. This mechanism might be responsible to tame uncontrolled receptor field growth. Combining these results with developmental GluR-IIA dynamics leads to a comprehensive physiological concept, where Hebbian plasticity guides growth of postsynaptic receptor fields and sparse transmitter release stabilises receptor fields by preventing overgrowth.
Additionally, a novel mechanism of retrograde signaling was discovered, where direct postsynaptic channelrhodopsin-2 based stimulation, without involvement of presynaptic neurotransmitter release, leads to presynaptic depression. This phenomenon is reminiscent of a known retrograde homeostatic mechanism, of inverted polarity, where neurotransmitter release is upregulated, upon reduction of postsynaptic sensitivity.
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.
Memory is dynamic: shortly after acquisition it is susceptible to amnesic treatments, gets gradually consolidated, and becomes resistant to retrograde amnesia (McGaugh, 2000). Associative olfactory memory of the fruit fly Drosophila melanogaster also shows these features. After a single associative training where an odor is paired with electric shock (Quinn et al., 1974; Tully and Quinn, 1985), flies form an aversive odor memory that lasts for several hours, consisting of qualitatively different components. These components can be dissociated by mutations, their underlying neuronal circuitry and susceptibility to amnesic treatments (Dubnau and Tully, 1998; Isabel et al., 2004; Keene and Waddell, 2007; Masek and Heisenberg, 2008; Xia and Tully, 2007). A component that is susceptible to an amnesic treatment, i.e. anesthesia-sensitive memory (ASM), dominates early memory, but decays rapidly (Margulies et al., 2005; Quinn and Dudai, 1976). A consolidated anesthesia-resistant memory component (ARM) is built gradually within the following hours and lasts significantly longer (Margulies et al., 2005; Quinn and Dudai, 1976). I showed here that the establishment of ARM requires less intensity of shock reinforcement than ASM. ARM and ASM rely on different molecular and/or neuronal processes: ARM is selectively impaired in the radish mutant, whereas for example the amnesiac and rutabaga genes are specifically required for ASM (Dudai et al., 1988; Folkers et al., 1993; Isabel et al., 2004; Quinn and Dudai, 1976; Schwaerzel et al., 2007; Tully et al., 1994). The latter comprise the cAMP signaling pathway in the fly, with the PKA being its supposed major target (Levin et al., 1992). Here I showed that a synapsin null-mutant encoding the evolutionary conserved phosphoprotein Synapsin is selectively impaired in the labile ASM. Further experiments suggested Synapsin as a potential downstream effector of the cAMP/PKA cascade. Similar to my results, Synapsin plays a role for different learning tasks in vertebrates (Gitler et al., 2004; Silva et al., 1996). Also in Aplysia, PKA-dependent phosphorylation of Synapsin has been proposed to be involved in regulation of neurotransmitter release and short-term plasticity (Angers et al., 2002; Fiumara et al., 2004). Synapsin is associated with a reserve pool of vesicles at the presynapse and is required to maintain vesicle release specifically under sustained high frequency nerve stimulation (Akbergenova and Bykhovskaia, 2007; Li et al., 1995; Pieribone et al., 1995; Sun et al., 2006). In contrast, the requirement of Bruchpilot, which is homologous to the mammalian active zone proteins ELKS/CAST (Wagh et al., 2006), is most pronounced in immediate vesicle release (Kittel et al., 2006). Under repeated stimulation of a bruchpilot mutant motor neuron, immediate vesicle release is severely impaired whereas the following steady-state release is still possible (Kittel et al., 2006). In line with that, knockdown of the Bruchpilot protein causes impairment in clustering of Ca2+ channels to the active zones and a lack of electron-dense projections at presynaptic terminals (T-bars). Thus, less synaptic vesicles of the readily-releasable pool are accumulated to the release sites and their release probability is severely impaired (Kittel et al., 2006; Wagh et al., 2006). First, I showed that Bruchpilot is required for aversive olfactory memory and localized the requirement of Bruchpilot to the Kenyon cells of the mushroom body, the second-order olfactory interneurons in Drosophila. Furthermore, I demonstrated that Bruchpilot selectively functions for the consolidated anesthesia-resistant memory. Since Synapsin is specifically required for the labile anesthesia sensitive memory, different synaptic proteins can dissociate consolidated and labile components of olfactory memory and two different modes of neurotransmission (high- vs. low frequency dependent) might differentiate ASM and ARM.
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.
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.
Since the fruit fly Drosophila melanogaster entered the laboratories as a model organism, new genetic, physiological, molecular and behavioral techniques for the functional analysis of the brain rapidly accumulated. Nowadays this concerted assault obtains its main thrust form Gal4 expression patterns that can be visualized and provide the means for manipulating -in unrestrained animals- groups of neurons of the brain. To take advantage of these patterns one needs to know their anatomy. This thesis describes the Virtual Insect Brain (VIB) protocol, a software package for the quantitative assessment, comparison, and presentation of neuroanatomical data. It is based on the 3D-reconstruction and visualization software Amira (Mercury Inc.). Its main part is a standardization procedure which aligns individual 3D images (series of virtual sections obtained by confocal microscopy) to a common coordinate system and computes average intensities for each voxel (volume pixel). The VIB protocol facilitates direct comparison of gene expression patterns and describes their interindividual variability. It provides volumetry of brain regions and helps to characterize the phenotypes of brain structure mutants. Using the VIB protocol does not require any programming skills since all operations are carried out at a (near to) self-explanatory graphical user interface. Although the VIB protocol has been developed for the standardization of Drosophila neuroanatomy, the program structure can be used for the standardization of other 3D structures as well. Standardizing brains and gene expression patterns is a new approach to biological shape and its variability. Using the VIB protocol consequently may help to integrate knowledge on the correlation of form and function of the insect brain. The VIB protocol provides a first set of tools supporting this endeavor in Drosophila. The software is freely available at http://www.neurofly.de.
Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis.
Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm.
Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels.
At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites.
Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation.
Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation.
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.
Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla’s edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors’ are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons’ conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs’ terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations
The correct regulation of cell growth and proliferation is essential during normal animal development. Myc proteins function as transcription factors, being involved in the con-trol of many growth- and proliferation-associated genes and deregulation of Myc is one of the main driving factors of human malignancies.
The first part of this thesis focuses on the identification of directly regulated Myc target genes in Drosophila melanogaster, by combining ChIPseq and RNAseq approaches. The analysis results in a core set of Myc target genes of less than 300 genes which are mainly involved in ribosome biogenesis. Among these genes we identify a novel class of Myc targets, the non-coding small nucleolar RNAs (snoRNAs). In vivo studies show that loss of snoRNAs not only impairs growth during normal development, but that overexpression of several snoRNAs can also enhance tumor development in a neu-ronal tumor model. Together the data show that Myc acts as a master regulator of ribo-some biogenesis and that Myc’s transforming effects in tumor development are at least partially mediated by the snoRNAs.
In the second part of the thesis, the interaction of Myc and the Zf-protein Chinmo is described. Co-immunoprecipitations of the two proteins performed under endogenous and exogenous conditions show that they interact physically and that neither the two Zf-domains nor the BTB/POZ-domain of Chinmo are important for this interaction. Fur-thermore ChIP experiments and Myc dependent luciferase assays show that Chinmo and Myc share common target genes, and that Chinmo is presumably also involved in their regulation. While the exact way of how Myc and Chinmo genetically interact with each other still has to be investigated, we show that their interaction is important in a tumor model. Overexpression of the tumor-suppressors Ras and Chinmo leads to tu-mor formation in Drosophila larvae, which is drastically impaired upon loss of Myc.
Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis.
In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbm’s RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbm’s RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts – Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation.
Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingers’ functions.
In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions.
For a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures.
For a large fraction of the proteins expressed in the human brain only the primary
structure is known from the genome project. Proteins conserved in evolution can
be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal
antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and
characterized with the aim to identify the target antigen. The mAb ab52 was found
to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western
blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a
single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15
(epidermal growth factor receptor pathway substrate clone 15) to be a strong
candidate. Another mAb from the library, aa2, was already found to recognize
EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and
2D electrophoretic separations revealed similar patterns, hence indicating that both
antigens could represent the same protein. Finally absence of the wild-type signal
in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52
antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila
homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for
applications like immunoprecipitation (IP). It has already been submitted to the
Developmental Studies Hybridoma Bank (DSHB) to be easily available for the
entire research community.
The mAb na21 was also found to be an IgM. It recognizes a membrane associated
antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature
of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of
the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to
biochemically purify the endogenously expressed protein from the tissue, gave
99
promising results but could not be completed due to lack of time. Thus
biochemical purification of the protein seems possible in order to facilitate its
identification by mass spectrometry. Several other mAbs were studied for their
staining pattern on cryosections and whole mounts of Drosophila brains. However,
many of these mAbs stained very few structures in the brain, which indicated that
only a very limited amount of protein would be available as starting material.
Because these antibodies did not produce signals on Western blots, which made it
impossible to enrich the antigens by electrophoretic methods, we did not attempt
their purification. However, the specific localization of these proteins makes them
highly interesting and calls for their further characterization, as they may play a
highly specialized role in the development and/or function of the neural circuits
they are present in. The purification and identification of such low expression
proteins would need novel methods of enrichment of the stained structures.
Functional and genetic dissection of mechanosensory organs of \(Drosophila\) \(melanogaster\)
(2016)
In Drosophila larvae and adults, chordotonal organs (chos) are highly versatile mechanosensors
that are essential for proprioception, touch sensation and hearing. Chos share molecular,
anatomical and functional properties with the inner ear hair cells of mammals. These multiple
similarities make chos powerful models for the molecular study of mechanosensation.
In the present study, I have developed a preparation to directly record from the sensory neurons
of larval chos (from the lateral chos or lch5) and managed to correlate defined mechanical inputs
with the corresponding electrical outputs. The findings of this setup are described in several case
studies.
(1) The basal functional lch5 parameters, including the time course of response during continuous
mechanical stimulation and the recovery time between successive bouts of stimulation, was
characterized.
(2) The calcium-independent receptor of α-latrotoxin (dCIRL/Latrophilin), an Adhesion class G
protein-coupled receptor (aGPCR), is identified as a modulator of the mechanical signals
perceived by lch5 neurons. The results indicate that dCIRL/Latrophilin is required for the
perception of external and internal mechanical stimuli and shapes the sensitivity of neuronal
mechanosensation.
(3) By combining this setup with optogenetics, I have confirmed that dCIRL modulates lch5
neuronal activity at the level of their receptor current (sensory encoding) rather than their ability
to generate action potentials.
(4) dCIRL´s structural properties (e.g. ectodomain length) are essential for the mechanosensitive
properties of chordotonal neurons.
(5) The versatility of chos also provides an opportunity to study multimodalities at multiple levels.
In this context, I performed an experiment to directly record neuronal activities at different
temperatures. The results show that both spontaneous and mechanically evoked activity increase
in proportion to temperature, suggesting that dCIRL is not required for thermosensation in chos.
These findings, from the development of an assay of sound/vibration sensation, to neuronal
signal processing, to molecular aspects of mechanosensory transduction, have provided the first
insights into the mechanosensitivity of dCIRL.
In addition to the functional screening of peripheral sensory neurons, another
electrophysiological approach was applied in the central nervous system: dCIRL may impact the
excitability of the motor neurons in the ventral nerve cord (VNC). In the second part of my work,
whole-cell patch clamp recordings of motor neuron somata demonstrated that action potential
firing in the dCirl\(^K\)\(^O\) did not differ from control samples, indicating comparable membrane
excitability.
Since Channelrhodopsins has been described first and introduced successfully in freely moving animals (Nagel et al., 2003 and 2005), tremendous impact has been made in this interesting field of neuroscience. Subsequently, many different optogenetic tools have been described and used to address long-lasting scientific issues. Furthermore, beside the ‘classical’ Channelrhodopsin-2 (ChR2), basically a cation-selective ion channel, also altered ChR2 descendants, anion selective channels and light-sensitive metabotropic proteins have expanded the optogenetic toolbox. However, in spite of this variety of different tools most researches still pick Channelrhodopsin-2 for their optogenetic approaches due to its well-known kinetics. In this thesis, an improved Channelrhodopsin, Channelrhodopsin2-XXM (ChR2XXM), is described, which might become an useful tool to provide ambitious neuroscientific approaches by dint of its characteristics. Here, ChR2XXM was chosen to investigate the functional consequences of Drosophila larvae lacking latrophilin in their chordotonal organs. Finally, the functionality of GtACR, was checked at the Drosophila NMJ. For a in-depth characterisation, electrophysiology along with behavioural setups was employed. In detail, ChR2XXM was found to have a better cellular expression pattern, high spatiotemporal precision, substantial increased light sensitivity and improved affinity to its chromophore retinal, as compared to ChR2. Employing ChR2XXM, effects of latrophilin (dCIRL) on signal transmission in the chordotonal organ could be clarified with a minimum of side effects, e.g. possible heat response of the chordotonal organ, due to high light sensitivity. Moreover, optogenetic activation of the chordotonal organ, in vivo, led to behavioural changes. Additionally, GtACR1 was found to be effective to inhibit motoneuronal excitation but is accompanied by unexpected side effects. These results demonstrate that further improvement and research of optogenetic tools is highly valuable and required to enable researchers to choose the best fitting optogenetic tool to address their scientific questions.
The transcription factor Myc interacts with several co-factors to regulate growth and proliferationand thereby enables normal animal development. Deregulation of Myc is associated witha wide range of human tumors. Myc binds to DNA together with its dimerization partner Max, preferentially to canonical E-box motifs, but this sequence-specific interaction is probably not sufficient for Myc’s binding to target genes.
In this work, the PAF1 complex was characterized as a novel co-factor of Myc in Drosophila melanogaster. All components of the complex are required for Myc’s recruitment to chromatin, but the subunit Atu has the strongest effect on Myc's binding to target genes through ist direct physical interaction with Myc. Unexpectedly, the impact of Atu depletion on the Expression of Myc target genes was weak compared to its effect on Myc binding. However, the influence of Atu becomes more prominent in situations of elevated Myc levels in vivo . Mycrepressed as well as Myc-activated targets are affected, consistent with the notion that Myc
recruitment is impaired.
An independent set of analyses revealed that Myc retains substantial activity even in the complete absence of Max. The overexpression of Myc in Max0 mutants specifically blocks their pupariation without affecting their survival, which raised the possibility that Myc might
affect ecdysone biosynthesis. This connection was studied in the second part of this Thesis which showed that Myc inhibits the expression of ecdysteroidogenic genes and thereby the production of ecdysone. Myc most likely affects the signaling pathways (PTTH and insulin
signaling) upstream of the PG, the organ where ecdysone is produced. By combining existing ChIPseq, RNAseq and electronic annotation data, we identified five potential Maxindependent Myc targets and provided experimental data that they might be involved in Myc's effect on Max mutant animals. Together our data confirm that some Myc functions are Max-independent and they raise the possibility that this effect might play a role during replication.
Latrophilin, alternatively named calcium-independent receptor of α-latrotoxin (CIRL), resembles a prototype of the adhesion class G-protein coupled receptors (GPCRs). Initially identified as a high-affinity receptor for α-latrotoxin, a component of the black widow spider, latrophilins are now associated with various distinct functions, such as synaptic exocytosis, tissue polarity and fertility (Tobaben et al., 2002; Langenhan et al., 2009; Promel et al., 2012). Despite these exploratory efforts the precise subcellular localisation as well as the endogenous ligand of CIRL still remains elusive. In this work genetic experiments, imaging approaches and behavioural studies have been used to unravel the localisation and physiological function of the latrophilin homolog dCirl in Drosophila melanogaster. Containing only one latrophilin homolog together with its genetic accessibility and well-established transgenic approaches, Drosophila seemed an ideally suited model organism. The present study showed that dCirl is widely expressed in the larval central nervous system including moto- and sensory neurons. Further, this work revealed that removal of the latrophilin homolog does not greatly affect synaptic transmission but it seems that aspects of the postsynaptic structural layout are controlled by dCIRL in the fruit fly. Additionally, dCirl expression at the transcriptional level was confirmed in larval and adult chordotonal organs, specialised mechanosensors implicated in proprioception (Eberl, 1999). Expression of dCIRL at the protein level could not yet been confirmed in moto- and sensory neurons likely due to low endogenous expression. However, behavioural studies using dCirl knockout mutant larvae indicated a putative mechanosensory function of dCIRL regarding touch sensitivity and locomotion behaviour.
The second part of this thesis presents a strategy to examine interactions between several presynaptic proteins in living cells. The attempt described in this work is based on the discovery that GFP when split into two non-fluorescent fragments can form a fluorescent complex. The association of the fragments can be facilitated by fusing them to two proteins that interact with each other. Therefore, the split GFP method enables direct visualization of synaptic protein interactions in living cells. In initial experiments I could show that full length reporter protein fusions with n-Synaptobrevin (n-Syb), Synaptotagmin (Syt) and Syntaxin (Syx) allow expression in Drosophila and confirmed that fusion to either end of each synaptic protein did not impair expression or influence the viability of transgenic flies. Further, transgenes containing protein fusions of Syx, Syt, and n-Syb with split GFP fragments were established in previous studies (Gehring, 2010). The present work characterises the interaction of these protein fusions during different stages of synaptic vesicle turnover at active zones such as synaptic vesicle docking at the presynaptic membrane and vesicle fusion. These results suggest that the spGFP assay seems only partly suitable for resolving fast and transient protein-protein interactions at larval Drosophila active zones in vivo.
To unravel the role of single genes underlying certain biological processes, scientists often use amorphic or hypomorphic alleles. In the past, such mutants were often created by chance. Enormous approaches with many animals and massive screening effort for striking phenotypes were necessary to find a needle in the haystack. Therefore at the beginning chemical mutagens or radiation were used to induce mutations in the genome. Later P-element insertions and inaccurate jump-outs enabled the advantage of potential larger deletions or inversions. The mutations were characterized and subsequently kept in smaller populations in the laboratories. Thus additional mutations with unknown background effects could accumulate.
The precision of the knockout through homologous recombination and the additional advantage of being able to generate many useful rescue constructs that can be easily reintegrated into the target locus made us trying an ends-out targeting procedure of the two core clock genes period and timeless in Drosophila melanogaster. Instead of the endogenous region, a small fragment of approximately 100 base pairs remains including an attP-site that can be used as integration site for in vitro created rescue constructs. After a successful ends-out targeting procedure, the locus will be restored with e.g. flies expressing the endogenous gene under the native promoter at the original locus coupled to a fluorescence tag or expressing luciferase.
We also linked this project to other research interests of our work group, like the epigenetic related ADAR-editing project of the Timeless protein, a promising newly discovered feature of time point specific timeless mRNA modification after transcription with yet unexplored consequences. The editing position within the Timeless protein is likewise interesting and not only noticed for the first time. This will render new insights into the otherwise not-satisfying investigation and quest for functional important sequences of the Timeless protein, which anyway shows less homology to other yet characterized proteins.
Last but not least, we bothered with the question of the role of Shaggy on the circadian clock. The impact of an overexpression or downregulation of Shaggy on the pace of the clock is obvious and often described. The influence of Shaggy on Period and Timeless was also shown, but for the latter it is still controversially discussed. Some are talking of a Cryptochrome stabilization effect and rhythmic animals in constant light due to Shaggy overexpression, others show a decrease of Cryptochrome levels under these conditions. Also the constant light rhythmicity of the flies, as it was published, could not be repeated so far. We were able to expose the conditions behind the Cryptochrome stabilization and discuss possibilities for the phenomenon of rhythmicity under constant light due to Shaggy overexpression.
In this thesis I studied psychological aspects in the behaviour of Drosophila, and especially Drosophila larvae. After an introduction where I present the general scientific context and describe the mechanisms of olfactory perception as well as of classical and operant conditioning, I present the different experiments that I realised during my PhD. Perception The second chapter deals with the way adult Drosophila generalise between single odours and binary mixtures of odours. I found that flies perceive a mixture of two odours as equally similar to the two elements composing it; and that the intensity as well as the physico-chemical nature of the elements composing a mixture affect the degree of generalisation between this mixture and one of its elements. These findings now call for further investigation on the physiological level, using functional imaging. Memory The third chapter presents a series of experiments in Drosophila larvae in order to define some characteristics of a new protocol for classical aversive learning which involves associating odours with mechanical disturbance as a punishment. The protocol and the first results should open new doors for the study of classical conditioning in Drosophila larvae, by allowing the comparison between two types of aversive memory (gustatory vs. mechanical reinforcement), including a comparison of their neurogenetic bases. It will also allow enquiries into the question whether these respective memories are specific for the kind of reinforcer used. Agency The fourth chapter documents our attempts to establish operant memory in Drosophila larvae. By analysing the first moments of the test, I could reveal that the larvae modified their behaviour according to their previous operant training. However, this memory seems to be quickly extinguished during the course of the test. We now aim at repeating these results and improving the protocol, in order to be able to systematically study the mechanisms allowing and underlying operant learning in Drosophila larvae. In the fifth chapter, I use the methods developed in chapter four for an analysis of larval locomotion. I determine whether larval locomotion in terms of speed or angular speed is affected by a treatment with the “cognitive enhancer” Rhodiola rosea, or by mutations in the Synapsin or SAP47 genes which are involved in the formation of olfactory memory. I also characterize the modifications induced by the presence of gustatory stimuli in the substrate on which the larvae are crawling. This thesis thus brings new elements to the current knowledge of Drosophila
Learning and memory is considered to require synaptic plasticity at presynaptic specializations of neurons. Kenyon cells are the intrinsic neurons of the primary olfactory learning center in the brain of arthropods – the mushroom body neuropils. An olfactory mushroom body memory trace is supposed to be located at the presynapses of Kenyon cells. In the calyx, a sub-compartment of the mushroom bodies, Kenyon cell dendrites receive olfactory input provided via projection neurons. Their output synapses, however, were thought to reside exclusively along their axonal projections outside the calyx, in the mushroom body lobes. By means of high-resolution imaging and with novel transgenic tools, we showed that the calyx of the fruit fly Drosophila melanogaster also comprised Kenyon cell presynapses. At these presynapses, synaptic vesicles were present, which were capable of neurotransmitter release upon stimulation. In addition, the newly identified Kenyon cell presynapses shared similarities with most other presynapses: their active zones, the sites of vesicle fusion, contained the proteins Bruchpilot and Syd-1. These proteins are part of the cytomatrix at the active zone, a scaffold controlling synaptic vesicle endo- and exocytosis. Kenyon cell presynapses were present in γ- and α/β-type KCs but not in α/β-type Kenyon cells.
The newly identified Kenyon cell derived presynapses in the calyx are candidate sites for an olfactory associative memory trace. We hypothesize that, as in mammals, recurrent neuronal activity might operate for memory retrieval in the fly olfactory system.
Moreover, we present evidence for structural synaptic plasticity in the mushroom body calyx. This is the first demonstration of synaptic plasticity in the central nervous system of Drosophila melanogaster. The volume of the mushroom body calyx can change according to changes in the environment. Also size and numbers of microglomeruli - sub-structures of the calyx, at which projection neurons contact Kenyon cells – can change. We investigated the synapses within the microglomeruli in detail by using new transgenic tools for visualizing presynaptic active zones and postsynaptic densities. Here, we could show, by disruption of the projection neuron - Kenyon cell circuit, that synapses of microglomeruli were subject to activity-dependent synaptic plasticity. Projection neurons that could not generate action potentials compensated their functional limitation by increasing the number of active zones per microglomerulus. Moreover, they built more and enlarged microglomeruli. Our data provide clear evidence for an activity-induced, structural synaptic plasticity as well as for the activity-induced reorganization of the olfactory circuitry in the mushroom body calyx.
Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication.
Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains.
This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.
The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication.
Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature.
Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs).
The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day.
To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner.
However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock.
Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.
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.