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Eine der größten Herausforderungen in der Neurobiologie ist es, die neuronalen Prozesse zu verstehen, die Lernen und Gedächtnis zugrundeliegen. Welche biochemischen Pfade liegen z.B. der Koinzidenzdetektion von Reizen (klassische Konditionierung) oder einer Handlung und ihren Konsequenzen (operante Konditionierung) zugrunde? In welchen neuronalen Unterstrukturen werden diese Informationen gespeichert? Wie ähnlich sind die Stoffwechselwege, die diese beiden Arten des assoziativen Lernens vermitteln und auf welchem Niveau divergieren sie? Drosophila melanogaster ist wegen der Verfügbarkeit von Lern-Paradigmen und neurogenetischen Werkzeugen ein geeigneter Modell-Organismus, zum diese Fragen zu adressieren. Er ermöglicht eine umfangreiche Studie der Funktion des Gens S6KII, das in der Taufliege in klassischer und operanter Konditionierung unterschiedlich involviert ist (Bertolucci, 2002; Putz et al., 2004). Rettungsexperimenten zeigen, dass die olfaktorische Konditionierung in der Tully Maschine (ein klassisches, Pawlow’sches Konditionierungsparadigma) von dem Vorhandensein eines intakten S6KII Gens abhängt. Die Rettung war sowohl mit einer vollständigen, als auch einer partiellen Deletion erfolgreich und dies zeigt, dass der Verlust der phosphorylierenden Untereinheit der Kinase die Hauptursache des Funktionsdefektes war. Das GAL4/UAS System wurde benutzt, um die S6KII Expression zeitlich und räumlich zu steuern. Es wurde gezeigt, dass die Expression der Kinase während des adulten Stadiums für die Rettung hinreichend war. Dieser Befund schließt eine Entwicklungsstörung als Ursache für den mutanten Phänotyp aus. Außerdem zeigte die gezielte räumliche Rettung von S6KII die Notwendigkeit der Pilzkörper und schloss Strukturen wie das mediane Bündel, die Antennalloben und den Zentralkomplex aus. Dieses Muster ist dem vorher mit der rutabaga Mutation identifizierten sehr ähnlich (Zars et al., 2000). Experimente mit der Doppelmutante rut, ign58-1 deuten an, dass rutabaga und S6KII im gleichen Signalweg aktiv sind. Vorhergehende Studien hatten bereits gezeigt, dass die unterschiedlichen Ergebnisse bei operanter und klassischer Konditionierung auf verschiedenen Rollen für S6KII in den zwei Arten des Lernens hindeuten (Bertolucci, 2002; Putz, 2002). Diese Schlussfolgerung wurde durch den mutanten Phänotyp der transgenen Linien in der Positionskonditionierung und ihr wildtypisches Verhalten in der klassischen Konditionierung zusätzlich bekräftigt. Eine neue Art von Lern-Experiment, genannt „Idle Experiment“, wurde entworfen. Es basiert auf der Konditionierung der Laufaktivität, stellt eine operante Aufgabenstellung dar und überwindet einige der Limitationen des „Standard“ Heat-Box Experimentes. Die neue Art des Idle Experimentes erlaubt es, „gelernte Hilflosigkeit“ in Fliegen zu erforschen, dabei zeigte sich eine erstaunliche Ähnlichkeit zu den Vorgängen in komplizierteren Organismen wie Ratten, Mäusen oder Menschen. Gelernte Hilflosigkeit in der Taufliege wurde nur in den Weibchen beobachtet und wird von Antidepressiva beeinflusst.
In this thesis, synaptic transmission was studied electrophysiologically at an invertebrate model synapse, the neuromuscular junction of the Drosophila 3rd instar wandering larvae. In the first part, synaptic function is characterized at the neuromuscular junction in fly lines which are null mutants for the synaptic proteins “the synapse associated protein of 47 kDa” (Sap-47156), Synapsin (Syn97), the corresponding double mutant (Sap-47156, Syn97), a null mutant for an as yet uncharacterized Drosophila SR protein kinase, the Serine-Arginine protein kinase 3 (SRPK3), and the Löchrig (Loe) mutant which shows a strong neurodegenerative phenotype. Intracellular voltage recordings from larval body wall muscles 6 and 7 were performed to measure amplitude and frequency of spontaneous single vesicle fusion events (miniature excitatory junction potentials or mEJPs). Evoked excitatory junction potentials (eEJPs) at different frequencies and calcium concentrations were also measured to see if synaptic transmission was altered in mutants which lacked these synaptic proteins. In addition, structure and morphology of presynaptic boutons at the larval neuromuscular junction were examined immunohistochemically using monoclonal antibodies against different synaptic vesicle proteins (SAP-47, CSP, and Synapsin) as well as the active zone protein Bruchpilot. Synaptic physiology and morphology was found to be similar in all null mutant lines. However, Löchrig mutants displayed an elongated bouton morphology, a significant shift towards larger events in mEJP amplitude frequency histograms, and increased synaptic facilitation during a 10 Hz tetanus. These deficits suggest that Loe mutants may have a defect in some aspect of synaptic vesicle recycling. The second part of this thesis involved the electrophysiological characterization of heterologously expressed light activated proteins at the Drosophila neuromuscular junction. Channelrhodopsin-2 (ChR2), a light gated ion channel, and a photoactivated adenylate cyclase (PAC) were expressed in larval motor neurons using the UAS-Gal4 system. Single EJPs could be recorded from muscles 15, 16, and 17 when larva expressing ChR2 were illuminated with short (100 ms) light pulses, whereas long light pulses (10 seconds) resulted in trains of EJPs with a frequency of around 25 Hz. Larva expressing PAC in preparations where motor neurons were cut from the ventral ganglion displayed a significant increase in mEJP frequency after a 1 minute exposure to blue light. Evoked responses in low (.2 mM) calcium were also significantly increased when PAC was stimulated with blue light. When motor nerves were left intact, PAC stimulation resulted in light evoked EJPs in muscles 6 and 7 in a manner consistent with RP3 motor neuron activity. ChR2 and PAC are therefore useful and reliable tools for manipulating neuronal activity in vivo.
Past experience contributes to behavioural organization mainly via learning: Animals learn otherwise ordinary cues as predictors for biologically significant events. This thesis studies such predictive, associative learning, using the fruit fly Drosophila melanogaster. I ask two main questions, which complement each other: One deals with the processing of those cues that are to be learned as predictors for an important event; the other one deals with the processing of the important event itself, which is to be predicted. Do fruit flies learn about combinations of olfactory and visual cues? I probe larval as well as adult fruit flies for the learning about combinations of olfactory and visual cues, using a so called ‘biconditional discrimination’ task: During training, one odour is paired with reinforcement only in light, but not in darkness; the other odour in turn is reinforced only in darkness, but not in light. Thus, neither the odours nor the visual conditions alone predict reinforcement, only combinations of both do. I find no evidence that either larval or adult fruit flies were to solve such task, speaking against a cross-talk between olfactory and visual modalities. Previous studies however suggest such cross-talk. To reconcile these results, I suggest classifying different kinds of interaction between sensory modalities, according to their site along the sensory-motor continuum: I consider an interaction ‘truly’ cross-modal, if it is between the specific features of the stimuli. I consider an interaction ’amodal’ if it instead engages the behavioural tendencies or ‘values’ elicited by each stimulus. Such reasoning brings me to conclude that different behavioural tasks require different kinds of interaction between sensory modalities; whether a given kind of interaction will be found depends on the neuronal infrastructure, which is a function of the species and the developmental stage. Predictive learning of pain-relief in fruit flies Fruit flies build two opposing kinds of memory, based on an experience with electric shock: Those odours that precede shock during training are learned as predictors for punishment and are subsequently avoided; those odours that follow shock during training on the other hand are learned as signals for relief and are subsequently approached. I focus on such relief learning. I start with a detailed parametric analysis of relief learning, testing for reproducibility as well as effects of gender, repetition of training, odour identity, odour concentration and shock intensity. I also characterize how relief memories, once formed, decay. In addition, concerning the psychological mechanisms of relief learning, first, I show that relief learning establishes genuinely associative conditioned approach behaviour and second, I report that it is most likely not mediated by context associations. These results enable the following neurobiological analysis of relief learning; further, they will form in the future the basis for a mathematical model; finally, they will guide the researchers aiming at uncovering relief learning in other experimental systems. Next, I embark upon neurogenetic analysis of relief learning. First, I report that fruit flies mutant for the so called white gene build overall more ‘negative’ memories about an experience with electric shock. That is, in the white mutants, learning about the painful onset of shock is enhanced, whereas learning about the relieving offset of shock is diminished. As they are coherently affected, these two kinds of learning should be in a balance. The molecular mechanism of the effect of white on this balance remains unresolved. Finally, as a first step towards a neuronal circuit analysis of relief learning, I compare it to reward learning and punishment learning. I find that relief learning is distinct from both in terms of the requirement for biogenic amine signaling: Reward and punishment are respectively signalled by octopamine and dopamine, for relief learning, either of these seem dispensible. Further, I find no evidence for roles for two other biogenic amines, tyramine and serotonin in relief learning. Based on these findings I give directions for further research.
Morphologie und Organisation individueller oktopaminerger Neurone im Gehirn von Drosophila m.
(2009)
Das biogene Amin Oktopamin moduliert verschiedene Verhaltensweisen in Invertebraten. In verschiedenen Insektenspezies, wie Heuschrecken, Grillen oder Schaben, ist die Funktion und die Architektur des peripheren oktopaminergen Systems auf Einzelzellebene bekannt. Um die zelluläre Grundlage für die verschiedenen Funktionen von Oktopamin im Zentralnervensystem zu verstehen, ist eine detaillierte Analyse der Architektur des zentralen oktopaminergen Systems notwendig. Innerhalb meiner Doktorarbeit fertigte eine anatomische Karte individueller oktopaminerger Neurone des adulten Hirns von Drosophila an. Ich nutzte die Flp-out Technik, um einzelne oktopaminerge Neurone anzufärben. Anhand ihrer Projektionsmuster konnte ich 28 verschiedene Zelltypen in vier Oktopamin-immunoreaktiven Zellclustern identifizieren. Ihre Morphologie sowie die Verteilung genetischer Marker zeigte, dass die meisten Zelltypen mehrere Neuropile innervieren und dabei eine klare Trennung von Prä- und Postsynaptischen Regionen aufweisen. Die Mehrheit der Zelltypen bildet dendritische Verzweigungen in einer bestimmten Region, der posterioren Slope. Jedoch innerviert jeder Zelltyp stereotyp eine bestimmte Kombination von Zielregionen im Gehirn. Das deutet stark darauf hin, dass oktopaminerge Neurone kombinatorisch organisiert sind: Jedes individuelle Neuron scheint Komponente eines spezifischen neuronalen Schaltkreises zu sein. Dabei könnte jeder Zelltyp eine Art “Modul” darstellen, das selektiv bestimmte Funktionen in den jeweiligen Zielregionen moduliert. Das oktopaminerge Mittelliniencluster des Subösophagealen Ganglions zeigt eine besondere zelluläre Organisation. Es besteht aus gepaarten und ungepaarten Neuronen, die des Zentralgehirn mit extensiven Verzweigungen versorgen. Um die Ordnung hinter dieser komplexen Organisation zu verstehen, wurden die segmentale Organistion der Mittellinienneurone auf Einzelzellebene analysiert und ihre embryonalen Anlagen verglichen. Letzteres ermöglichte die morphologische Analyse von einzelnen oktopaminergen Mittellinienklonen. OA-VPM und OA-VUM Neurone bilden zusammen drei Subcluster im Subösophagealen Ganglion, die wahrscheinlich die drei gnathalen Neuromere repräsentieren. Alle OA-VUM Neurone stammen von der embryonalen Mittellinie ab. In den mandibularen und maxillaren Neuromeren formen sie morphologisch identische Zelltypen, mit stereotypen Innervationsmustern. OA-VPM Neurone gehen nicht aus der embryonalen Mittellinie hervor und sind nicht segmental dupliziert. Diese Arbeit vermittelt nicht nur einen Eindruck über die Architektur individueller oktopaminerger Neurone, sondern auch über die Organisation des oktopaminergen Systems auf Einzelzellebene.
Die Entwicklung von Ethanoltoleranz ist ein Indikator für eine mögliche Abhängigkeit von Alkohol. Der genaue molekulare Mechanismus der Ethanoltoleranzentwicklung ist jedoch nicht bekannt. Drosophila ermöglicht die molekulare und phänotypische Untersuchung von verschiedenen Mutanten mit veränderter Toleranz und kann so zu einem besseren Verständnis beitragen. Die hangAE10 Mutante entwickelt eine reduzierte Ethanoltoleranz, wobei dieser Phänotyp auf Defekte in der zellulären Stressantwort zurückzuführen ist. Für ein besseres Verständnis, in welchen molekularen Mechanismen bzw. Signalwegen HANG wirkt, wurde die Funktion des Proteins auf zellulärer Ebene analysiert und mögliche Zielgene charakterisiert. Die auffällige Proteinstruktur von HANG spricht für eine Interaktion mit Nukleinsäuren. Immunhistochemische Analysen von ektopisch exprimiertem Hangover Protein ergaben, dass dieses nicht mit der DNA co-lokalisiert und auch nicht an polytänen Chromosomen nachgewiesen werden kann. Die ektopische Expression von HANG in Speicheldrüsenzellen zeigte eine punktförmige Verteilung des Proteins innerhalb des Zellkerns. Dieses punktförmige Expressionsmuster wird häufig in RNA-bindenden Proteinen gefunden. Deshalb wurden Co-Lokalisationsstudien von HANG mit Markern für RNAmodifizierende Proteine durchgeführt. Dabei wurde keine Interaktion mit verschiedenen Markerproteinen des Spleißapparates gefunden. Mithilfe von in vitro Experimenten konnte aber die Bindung von RNA an bestimmten Hangover Proteinbereichen nachgewiesen werden Diese Ergebnisse legen nahe, dass HANG eine RNA-regulierende Funktion hat. In einem cDNA Microarray Experiment wurde das Gen dunce als mögliches Zielgen von Hangover identifiziert. Das Gen dunce kodiert für eine Phosphodiesterase, welche spezifisch cAMP hydrolysiert. Zur Bestätigung der cDNA Microarray Experimente wurden die dnc Transkriptunterschiede in Wildtyp und hangAE10 Mutante mithilfe von semiquantitativer RT-PCR für jede der vier Gruppen untersucht. Dabei konnte eine Reduktion der dncRMRA-Transkriptgruppe in hangAE10 Mutanten nachgewiesen werden. Aufgrund dieser Ergebnisse wurde die dncRMRA -spezifische dncΔ143 Mutante hergestellt und auf Verhaltensebene analysiert. Die Experimente zeigten, dass sowohl dnc1, als auch die dncΔ143 Mutante eine reduzierte Ethanoltoleranz und Defekte in der zellulären Stressantwort aufweisen. Für die Rettung der reduzierten Toleranz von hangAE10 und dncΔ143 in dncRMRA-spezifischen Neuronen wurde die dncRMRA Promotor- GAL4 Linie hergestellt. Die reduzierte Ethanoltoleranz der dncΔ143 Mutanten konnte über die Expression von UAS-dnc mit der dncRMRA-GAL4 Linie auf Wildtyp Level gerettet werden. Die reduzierte Toleranz der hangAE10 Mutante konnte mithilfe derselben GAL4 Linie verbessert werden. Dies beweist, dass in beiden Mutanten dieselben Zellen für die Entwicklung von Ethanoltoleranz benötigt werden und sie wahrscheinlich in der gleichen Signaltransduktionskaskade eine Funktion haben. Aufgrund der Anfälligkeit der UAS/ GAL4 Systems gegenüber Hitze war es außerdem nicht möglich die Defekte der zellulären Stressantwort von dncΔ143 bzw. hangAE10 Fliegen zu retten. Die Rettung der reduzierten Ethanoltoleranz der dcnΔ143 Mutante führte außerdem zu der Vermutung, dass die cAMP Regulation eine wichtige Funktion bei der Ethanoltoleranzentwicklung hat. Über die Expression von cAMP-regulierenden Proteinen in dncRMRA-spezifischen Neuronen wurde der Einfluss von cAMP bei Ethanol-induziertem Verhalten überprüft. Bei der Überexpression von dunce und rutabaga konnte weder eine Veränderung für die Ethanolsensitivität, noch für die Toleranzentwicklung festgestellt werden. Eine Erklärung hierfür wäre, dass Veränderungen in der cAMP Konzentration über Rückkopplungsmechanismen zwischen Dunce und Rutabaga ausgeglichen werden können. Für eine genauere Aussage müsste jedoch die cAMP Konzentration in diesen Fliegen gemessen werden. Die Überexpression von pka- in dncRMRA spezifischen Zellen führt zu einer erhöhten Ethanolresistenz. Das bedeutet, dass die Modulation der cAMP Konzentration durch dunce und rutabaga in dncRMRA spezifischen Zellen keinen Einfluss auf Ethanol-induziertes Verhalten hat, wohingegen die Stärke der cAMP vermittelten Signalverarbeitung über die cAMP-abhängige PKA zu Veränderungen im Verhalten führt. Für Mutanten des cAMP Signalweges ist außerdem bekannt, dass sie Defekte im olfaktorischen Lernen bzw. Gedächtnis aufweisen. Deshalb wurden die dncΔ143, dnc1 und hangAE10 Mutanten in diesem Paradigma getestet. Sowohl dnc1, als auch dncΔ143 Fliegen zeigten einen reduzierten Performance Index für das zwei und 30 Minuten Gedächtnis. Nach 180 Minuten verhielten sich die dncΔ143 Mutanten nicht mehr unterschiedlich zum Wildtyp, die dnc1 Mutante zeigte jedoch immer noch eine Reduktion des Performance Index im Vergleich zur Kontrolle. Demnach ist in dncΔ143 Mutanten nur das Kurzzeitgedächtnis betroffen, wohingegen hangAE10 Mutanten keine Reduktion des Performance Index für das olfaktorische Kurzzeitgedächtnis aufweisen. Die unterschiedlichen Ergebnisse der beiden Mutanten in der Gedächtnisentwicklung deuten außerdem daraufhin, dass Lernen und Gedächtnis in dncΔ143 und hangAE10 Mutanten von der Toleranzentwicklung unabhängig über unterschiedliche cAMP-abhängige Signaltransduktionskaskaden reguliert werden.
In dieser Arbeit wurden zwei Techniken zur Analyse der Funktion diverser Neuronen in Drosophila melanogaster angewendet. Im ersten Teil wurde mittels in-vivo Calcium Imaging Technik unter Verwendung des Calciumsensors Cameleon neuronale Aktivität entlang des olfaktorischen Signalweges registriert. Hierbei wurde die neuronale Repräsentation der Duftidentität und der Duftintensität untersucht. In Bezug auf diese Fragestellung wurde die Datenverarbeitung und Datenanalyse weiterentwickelt und standardisiert. Die Experimente führten zu dem Ergebnis, dass duftspezifische Aktivitätsmuster auf der Ebene des Antennallobus sehr gut unterscheidbar sind. Manche Aktivitätsmuster der präsentierten Düfte zeigten interessanterweise einen hohen Ähnlichkeitsgrad, wohingegen andere unähnlich waren. In höheren Gehirnzentren wie den Orten der terminalen Aborisationen der Projektionsneurone oder den Pilzkörper Kenyonzellen liegt eine starke Variabilität der duftevozierten Aktivitätsmuster vor, was generelle Interpretationen unmöglich macht und höchstens Vergleiche innerhalb eines Individuums zulässt. Des Weiteren konnte gezeigt werden, dass die Calciumsignale in den Rezeptorneuronen sowie prä- und postsynaptisch in den Projektionsneuronen bei Erhöhung der Konzentration der verschiedenen präsentierten Düfte über einen Bereich von mindestens drei Größenordnungen ansteigen. In den Kenyonzellen des Pilzkörper-Calyx und der Pilzkörper-Loben ist diese Konzentrationsabhängigkeit weniger deutlich ausgeprägt und im Falle der Loben nur für bestimmte Düfte detektierbar. Eine Bestätigung des postulierten „sparsed code“ der Duftpräsentation in den Pilzkörpern konnte in dieser Arbeit nicht erbracht werden, was möglicherweise daran liegt, dass eine Einzelzellauflösung mit der verwendeten Technik nicht erreicht werden kann. Im zweiten Teil dieser Arbeit sollte durch die Nutzung des lichtabhängigen Kationenkanals Channelrhodopsin-2 der Frage nachgegangen werden, ob bestimmte modulatorische Neurone die verstärkenden Eigenschaften eines bestrafenden oder belohnenden Stimulus vermitteln. Die lichtinduzierte Aktivierung von Channelrhodopsin-2 exprimierenden dopaminergen Neuronen als Ersatz für einen aversiven Reiz führte bei einer olfaktorischen Konditionierung bei Larven zur Bildung eines aversiven assoziativen Gedächtnisses. Im Gegensatz dazu induzierte die Aktivierung von Channelrhodopsin-2 in oktopaminergen/tyraminergen Neuronen als Ersatz für einen appetitiven Reiz ein appetitives assoziatives Gedächtnis. Diese Ergebnisse zeigen, dass dopaminerge Neurone bei Larven aversives Duftlernen, oktopaminerge/tyraminerge Neurone dagegen appetitives Duftlernen induzieren.
In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick & Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well.
Neuroanatomical data in fly brain research are mostly available as spatial gene expression patterns of genetically distinct fly strains. The Drosophila standard brain, which was developed in the past to provide a reference coordinate system, can be used to integrate these data. Working with the standard brain requires advanced image processing methods, including visualisation, segmentation and registration. The previously published VIB Protocol addressed the problem of image registration. Unfortunately, its usage was severely limited by the necessity of manually labelling a predefined set of neuropils in the brain images at hand. In this work I present novel tools to facilitate the work with the Drosophila standard brain. These tools are integrated in a well-known open-source image processing framework which can potentially serve as a common platform for image analysis in the neuroanatomical research community: ImageJ. In particular, a hardware-accelerated 3D visualisation framework was developed for ImageJ which extends its limited 3D visualisation capabilities. It is used for the development of a novel semi-automatic segmentation method, which implements automatic surface growing based on user-provided seed points. Template surfaces, incorporated with a modified variant of an active surface model, complement the segmentation. An automatic nonrigid warping algorithm is applied, based on point correspondences established through the extracted surfaces. Finally, I show how the individual steps can be fully automated, and demonstrate its application for the successful registration of fly brain images. The new tools are freely available as ImageJ plugins. I compare the results obtained by the introduced methods with the output of the VIB Protocol and conclude that our methods reduce the required effort five to ten fold. Furthermore, reproducibility and accuracy are enhanced using the proposed tools.
Synapsen als Stellen der Kommunikation zwischen Neuronen besitzen spezialisierte Bereiche – Aktive Zonen (AZs) genannt –, die aus einem hoch komplexen Netzwerk von Proteinen aufgebaut sind und die Maschinerie für den Prozess der Neurotransmitter-Ausschüttung und das Vesikel-Recycling beinhalten. In Drosophila ist das Protein Bruchpilot (BRP) ein wichtiger Baustein für die T-förmigen Bänder („T-Bars“) der präsynaptischen Aktiven Zonen. BRP ist notwendig für eine intakte Struktur der Aktiven Zone und eine normale Exocytose von Neurotransmitter-Vesikeln. Auf der Suche nach Mutationen, welche die Verteilung von Bruchpilot im Gewebe beeinträchtigen, wurde eine P-Element-Insertion im Gen CG11489 an der Position 79D identifiziert, welches eine Kinase kodiert, die einen hohen Grad an Homologie zur Familie der SR Proteinkinasen (SRPKs) von Säugern aufweist. Die Mitglieder dieser Familie zeichnen sich durch eine evolutionär hoch konservierte zweigeteilte Kinasedomäne aus, die durch eine nicht konservierte Spacer-Sequenz unterbrochen ist. SRPKs phosphorylieren SR-Proteine, die zu einer evolutionär hoch konservierten Familie Serin/Arginin-reicher Spleißfaktoren gehören und konstitutive sowie alternative Spleißprozesse steuern und damit auf post-transkriptioneller Ebene die Genexpression regulieren. Mutation des Srpk79D-Gens durch die P-Element-Insertion (Srpk79DP1) oder eine Deletion im Gen (Srpk79DVN Nullmutante) führt zu auffälligen BRP-Akkumulationen in larvalen und adulten Nerven. In der vorliegenden Arbeit wird gezeigt, dass diese BRP-Akkumulationen auf Ultrastruktur-Ebene ausgedehnten axonalen Agglomeraten elektronendichter Bänder entsprechen und von klaren Vesikeln umgeben sind. Charakterisierung durch Immuno-Elektronenmikroskopie ergab, dass diese Strukturen BRP-immunoreaktiv sind. Um die Bildung BRP-enthaltender Agglomerate in Axonen zu verhindern und damit eine intakte Gehirnfunktion zu gewährleisten, scheint die SRPK79D nur auf niedrigem Niveau exprimiert zu werden, da die endogene Kinase mit verschiedenen Antikörpern nicht nachweisbar war. Wie in anderen Arbeiten gezeigt werden konnte, ist die Expression der PB-, PC- oder PF-Isoform der vier möglichen SRPK79D-Varianten, die durch alternativen Transkriptionsstart in Exon eins beziehungsweise drei und alternatives Spleißen von Exon sieben zustande kommen, zur Rettung des Phänotyps der BRP-Akkumulation im Srpk79DVN Nullmutanten-Hintergrund ausreichend. Zur Charakterisierung der Rescue-Eigenschaften der SRPK79D-PE-Isoform wurde mit der Klonierung der cDNA in einen UAS-Vektor begonnen. Offenbar beruht die Bildung der axonalen BRP-Agglomerate nicht auf einer Überexpression von BRP in den betroffenen Neuronen, denn auch bei reduzierter Expression des BRP-Proteins im Srpk79DVN Nullmutanten-Hintergrund entstehen die BRP-Agglomerate. In Köpfen der Srpk79DVN Nullmutante ist die Gesamtmenge an Bruchpilot-Protein im Vergleich zum Wildtyp nicht deutlich verändert. Auch die auf Protein-Ebene untersuchte Expression der verschiedenen Isoformen der präsynaptischen Proteine Synapsin, Sap47 und CSP weicht in der Srpk79DVN Nullmutante nicht wesentlich von der Wildtyp-Situation ab, sodass sich keine Hinweise auf verändertes Spleißen der entsprechenden prä-mRNAs ergeben. Jedes der sieben bekannten SR-Proteine von Drosophila ist ein potentielles Zielprotein der SRPK79D. Knock-down-Experimente für die drei hier untersuchten SR-Proteine SC35, X16/9G8 und B52/SRp55 im gesamten Nervensystem durch RNA-Interferenz zeigten allerdings keinen Effekt auf die Verteilung von BRP im Gewebe. Hinsichtlich der Flugfähigkeit der Tiere hat die Srpk79DVN Nullmutation keinen additiven Effekt zum Knock-down des BRP-Proteins, denn die Doppelmutanten zeigten bei der Bestimmung des Anteils an flugunfähigen Tieren vergleichbare Werte wie die Einzelmutanten, die entweder die Nullmutation im Srpk79D-Gen trugen, oder BRP reduziert exprimierten. Vermutlich sind Bruchpilot und die SR Proteinkinase 79D somit Teil desselben Signalwegs. Durch Doppelfärbungen mit Antikörpern gegen BRP und CAPA-Peptide wurde abschließend entdeckt, dass Bruchpilot auch im Median- und Transvers-Nervensystem (MeN/TVN) von Drosophila zu finden ist, welche die Neurohämal-Organe beherbergen. Aufgabe dieser Organe ist die Speicherung und Ausschüttung von Neuropeptid-Hormonen. Daher ist zu vermuten, dass das BRP-Protein neben Funktionen bei der Neurotransmitter-Exocytose möglicherweise eine Rolle bei der Ausschüttung von Neuropeptiden spielt. Anders als in den Axonen der larvalen Segmental- und Intersegmentalnerven der Srpk79DVN Nullmutante, die charakteristische BRP-Agglomerate aufweisen, hat die Mutation des Srpk79D-Gens in den Axonen der Va-Neurone, die das MeN/TVN-System bilden, keinen sichtbaren Effekt auf die Verteilung von Brp, denn das Muster bei Färbung gegen BRP weist keine deutlichen Veränderungen zum Wildtyp auf.
This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.
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
An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade.
Understanding of complex interactions and events in a nervous system, leading from the molecular level up to certain behavioural patterns calls for interdisciplinary interactions of various research areas. The goal of the presented work is to achieve such an interdisciplinary approach to study and manipulate animal behaviour and its underlying mechanisms. Optical in vivo imaging is a new constantly evolving method, allowing one to study not only the local but also wide reaching activity in the nervous system. Due to ease of its genetic accessibility Drosophila melanogaster represents an extraordinary experimental organism to utilize not only imaging but also various optogenetic techniques to study the neuronal underpinnings of behaviour. In this study four genetically encoded sensors were used to investigate the temporal dynamics of cAMP concentration changes in the horizontal lobes of the mushroom body, a brain area important for learning and memory, in response to various physiological and pharmacological stimuli. Several transgenic lines with various genomic insertion sites for the sensor constructs Epac1, Epac2, Epac2K390E and HCN2 were screened for the best signal quality, one line was selected for further experiments. The in vivo functionality of the sensor was assessed via pharmacological application of 8-bromo-cAMP as well as Forskolin, a substance stimulating cAMP producing adenylyl cyclases. This was followed by recording of the cAMP dynamics in response to the application of dopamine and octopamine, as well as to the presentation of electric shock, odorants or a simulated olfactory signal, induced by acetylcholine application to the observed brain area. In addition the interaction between the shock and the simulated olfactory signal by simultaneous presentation of both stimuli was studied. Preliminary results are supporting a coincidence detection mechanism at the level of the adenylyl cyclase as postulated by the present model for classical olfactory conditioning. In a second series of experiments an effort was made to selecticvely activate a subset of neurons via the optogenetic tool Channelrhodopsin (ChR2). This was achieved by recording the behaviour of the fly in a walking ball paradigm. A new method was developed to analyse the walking behaviour of the animal whose brain was made optically accessible via a dissection technique, as used for imaging, thus allowing one to target selected brain areas. Using the Gal4-UAS system the protocerebral bridge, a substructure of the central complex, was highlighted by expressing the ChR2 tagged by fluorescent protein EYFP. First behavioural recordings of such specially prepared animals were made. Lastly a new experimental paradigm for single animal conditioning was developed (Shock Box). Its design is based on the established Heat Box paradigm, however in addition to spatial and operant conditioning available in the Heat Box, the design of the new paradigm allows one to set up experiments to study classical and semioperant olfactory conditioning, as well as semioperant place learning and operant no idleness experiments. First experiments involving place learning were successfully performed in the new apparatus.
Many organisms evolved an endogenous clock to adapt to the daily environmental changes caused by the earth’s rotation. Light is the primary time cue (“Zeitgeber”) for entrainment of circadian clocks to the external 24-h day. In Drosophila, several visual pigments are known to mediate synchronization to light: The blue-light photopigment Cryptochrome (CRY) and six well-described rhodopsins (Rh1-Rh6). CRY is present in the majority of clock neurons as well as in the compound eyes, whereas the location of rhodopsins is restricted to the photoreceptive organs – the compound eyes, the ocelli and the HB-eyelets. CRY is thought to represent the key photoreceptor of Drosophila’s circadian clock. Nevertheless, mutant flies lacking CRY (cry01) are able to synchronize their locomotor activity rhythms to light-dark (LD) cycles, but need significantly longer than wild-type flies. In this behavior, cry01 mutants strongly resemble mammalian species that do not possess any internal photoreceptors and perceive light information exclusively through their photoreceptive organs (eyes). Thus, a mammalian-like phase-shifting behavior would be expected in cry01 flies. We investigated this issue by monitoring a phase response curve (PRC) of cry01 and wild-type flies to 1-h light pulses of 1000 lux irradiance. Indeed, cry01 mutants produced a mammalian-similar so called type 1 PRC of comparatively low amplitude (< 25% of wild-type) with phase delays to light pulses during the early subjective night and phase advances to light pulses during the late subjective night (~1 h each). Despite the predominant role of CRY, the visual system contributes to the light sensitivity of the fly’s circadian clock, mainly around dawn and dusk. Furthermore, this phase shifting allows for the slow re-entrainment which we observed in cry01 mutants to 8-h phase delays of the LD 12 h:12 h cycle. However, cry01 also showed surprising differences in their shifting ability: First of all, their PRC was characterized by a second dead zone in the middle of the subjective night (ZT17-ZT19) in addition to the usual unresponsiveness during the subjective day. Second, in contrast to wild-type flies, cry01 mutants did not increase their shift of activity rhythms neither in response to longer stimuli nor to light pulses of higher irradiance. In contrast, both 6-h light pulses of 1000 lux and 1-h light pulses of 10,000 lux light intensity during the early subjective night even resulted in phase advances instead of the expected delays. Thus, CRY seems to be not only responsible for the high light sensitivity of the wild-type circadian clock, but is apparently also involved in integrating and processing light information. Rhodopsin 7 (Rh7) is a yet uncharacterized protein, but became a good photoreceptor candidate due to sequence similarities to the six known Drosophila Rhs. The second part of this thesis investigated the expression pattern of Rh7 and its possible functions, especially in circadian photoreception. Furthermore, we were interested in a potential interaction with CRY and thus, tested cry01 and rh70 cry01 mutants as well. Rh1 is the main visual pigment of the Drosophila compound eye and expressed in six out of eight photoreceptors cells (R1-R6) in each of the ~800 ommatidia. Motion vision depends exclusively on Rh1 function but, moreover, Rh1 plays an important structural role and assures proper photoreceptor cell development and maintenance. In order to investigate its possible photoreceptive function, we expressed Rh7 in place of Rh1. Rh7 was indeed able to overtake the role of Rh1 in both aspects: It prevented retinal degeneration and mediated the optomotor response (OR), a motion vision-dependent behavior. At the transcriptional level, rh7 is expressed at approximately equal amounts in adult fly brains and retinas. Due to a reduced specificity of anti-Rh7 antibodies, we could not verify this result at the protein level. However, analysis of rh7 null mutants (rh70) suggested different Rh7 functions in vivo. Previous experiments strongly indicated an increased sensitivity of the compound eyes in the absence of Rh7 and suggested impaired light adaptation. We aimed to test this hypothesis at the levels of circadian photoreception. Locomotor activity rhythms are a reliable output of the circadian clock. Rh70 mutant flies generally displayed a wild-type similar bimodal activity pattern comprising morning (M) and evening (E) activity bouts. Activity monitoring supported the proposed “shielding” function, since rh70 mutants behaved like wild-type flies experiencing high irradiances. Under all investigated conditions, their activity peaks lay further apart resulting in a prolonged midday break. The behavior of cry01 mutants was mainly characterized by an unexpectedly high flexibility in the timing of M and E activity bouts which allowed tracking of lights-on and lights-off even under extreme photoperiods. Activity profiles of the corresponding rh70 cry01 double mutants reflected neither synergistic nor antagonistic effects of Rh7 and CRY and were dominated by a broad E activity peak. In the future, the different circadian phenotypes will be further investigated on the molecular level by analysis of clock protein cycling in the underlying pacemaker neurons. The work of this thesis confirmed that Rh7 is indeed able to work as a photoreceptor and to initiate the classical phototransduction cascade. On the other hand, it provided further evidence at the levels of circadian photoreception that Rh7 might serve as a shielding pigment for Rh1 in vivo, thereby mediating proper light adaptation.
In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order “cortical” brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.
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.
Members of the Diaphanous (Dia) protein family are key regulators of fundamental actin driven cellular processes, which are conserved from yeast to humans. Researchers have uncovered diverse physiological roles in cell morphology, cell motility, cell polarity, and cell division, which are involved in shaping cells into tissues and organs. The identification of numerous binding partners led to substantial progress in our understanding of the differential functions of Dia proteins. Genetic approaches and new microscopy techniques allow important new insights into their localization, activity, and molecular principles of regulation.
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.
Large-scale anatomical and functional analyses of the connectivity in both invertebrate and mammalian brains have gained intense attention in recent years. At the same time, the understanding of synapses on a molecular level still lacks behind. We have only begun to unravel the basic mechanisms of how the most important synaptic proteins regulate release and reception of neurotransmitter molecules, as well as changes of synaptic strength. Furthermore, little is known regarding the stoichiometry of presynaptic proteins at different synapses within an organism. An assessment of these characteristics would certainly promote our comprehension of the properties of different synapse types. Presynaptic proteins directly influence, for example, the probability of neurotransmitter release as well as mechanisms for short-term plasticity. We have examined the strength of expression of several presynaptic proteins at different synapse types in the central nervous system of Drosophila melanogaster using immunohistochemistry. Clear differences in the relative abundances of the proteins were obvious on different levels: variations in staining intensities appeared from the neuropil to the synaptic level. In order to quantify these differences, we have developed a ratiometric analysis of antibody stainings. By application of this ratiometric method, we could assign average ratios of presynaptic proteins to different synapse populations in two central relays of the olfactory pathway. In this manner, synapse types could be characterized by distinct fingerprints of presynaptic protein ratios. Subsequently, we used the method for the analysis of aberrant situations: we reduced levels of Bruchpilot, a major presynaptic protein, and ablated different synapse or cell types. Evoked changes of ratio fingerprints were proportional to the modifications we had induced in the system. Thus, such ratio signatures are well suited for the characterization of synapses. In order to contribute to our understanding of both the molecular composition and the function of synapses, we also characterized a novel synaptic protein. This protein, Drep-2, is a member of the Dff family of regulators of apoptosis. We generated drep-2 mutants, which did not show an obvious misregulation of apoptosis. By contrast, Drep-2 was found to be a neuronal protein, highly enriched for example at postsynaptic receptor fields of the input synapses of the major learning centre of insects, the mushroom bodies. Flies mutant for drep-2 were viable but lived shorter than wildtypes. Basic synaptic transmission at both peripheral and central synapses was in normal ranges. However, drep-2 mutants showed a number of deficiencies in adaptive behaviours: adult flies were locomotor hyperactive and hypersensitive towards ethanol-induced sedation. Moreover, the mutant animals were heavily impaired in associative learning. In aversive olfactory conditioning, drep-2 mutants formed neither short-term nor anaesthesia-sensitive memories. We could demonstrate that Drep-2 is required in mushroom body intrinsic neurons for normal olfactory learning. Furthermore, odour-evoked calcium transients in these neurons, a prerequisite for learning, were reduced in drep-2 mutants. The impairment of the mutants in olfactory learning could be fully rescued by pharmacological application of an agonist to metabotropic glutamate receptors (mGluRs). Quantitative mass spectrometry of Drep-2 complexes revealed that the protein is associated with a large number of translational repressors, among them the fragile X mental retardation protein FMRP. FMRP inhibits mGluR-mediated protein synthesis. Lack of this protein causes the fragile X syndrome, which constitutes the most frequent monogenic cause of autism. Examination of the performance of drep-2 mutants in courtship conditioning showed that the animals were deficient in both short- and long-term memory. Drep-2 mutants share these phenotypes with fmrp and mGluR mutants. Interestingly, drep-2; fmrp double mutants exhibited normal memory. Thus, we propose a model in which Drep-2 antagonizes FMRP in the regulation of mGluR-dependent protein synthesis. Our hypothesis is supported by the observation that impairments in synaptic plasticity can arise if mGluR signalling is imbalanced in either direction. We suggest that Drep-2 helps in establishing this balance.