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Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling—the serotonin transporter–in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior.
The modulation of an animal’s behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. We here describe the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors.
Cryptochromes (CRYs) are a class of flavoproteins that sense blue light. In animals, CRYs are expressed in the eyes and in the clock neurons that control sleep/wake cycles and are implied in the generation and/or entrainment of circadian rhythmicity. Moreover, CRYs are sensing magnetic fields in insects as well as in humans. Here, we show that in the fruit fly Drosophila melanogaster CRY plays a light-independent role as “assembling” protein in the rhabdomeres of the compound eyes. CRY interacts with actin and appears to increase light sensitivity of the eyes by keeping the “signalplex” of the phototransduction cascade close to the membrane. By this way, CRY also enhances light-responses of the circadian clock.
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
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.
Swords are exaggerated male ornaments of swordtail fishes that have been of great interest to evolutionary biologists ever since Darwin described them in the Descent of Man (1871). They are a novel sexually selected trait derived from modified ventral caudal fin rays and are only found in the genus Xiphophorus. Another phylogenetically more widespread and older male trait is the gonopodium, an intromittent organ found in all poeciliid fishes, that is derived from a modified anal fin. Despite many evolutionary and behavioral studies on both traits, little is known so far about the molecular mechanisms underlying their development. By investigating transcriptomic changes (utilizing a RNA-Seq approach) in response to testosterone treatment in the swordtail fish, Xiphophorus hellerii, we aimed to better understand the architecture of the gene regulatory networks underpinning the development of these two evolutionary novelties. Large numbers of genes with tissue-specific expression patterns were identified. Among the sword genes those involved in embryonic organ development, sexual character development and coloration were highly expressed, while in the gonopodium rather more morphogenesis-related genes were found. Interestingly, many genes and genetic pathways are shared between both developing novel traits derived from median fins: the sword and the gonopodium. Our analyses show that a larger set of gene networks was co-opted during the development and evolution of the older gonopodium than in the younger, and morphologically less complex trait, the sword. We provide a catalog of candidate genes for future efforts to dissect the development of those sexually selected exaggerated male traits in swordtails.
Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Veränderungen ihrer Umwelt anzupassen. Während letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System täglich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltveränderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abhängigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gestörten biologischen Tagesrhythmen, wie zum Beispiel Schlafstörungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern kürzlich durchgeführte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine mögliche Verbindung zwischen Stress-induzierten und regulären rhythmischen Anpassungen des Organismus an Umweltveränderungen an. Molekulare und zelluläre Mechanismen dieser Verknüpfung sind bisher noch nicht bekannt.
Während die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine mögliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antikörperfärbungen sowie Studien mit Reporterlinien zeigen deutliche Färbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis für p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivität von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zusätzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht führen zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivität offenbaren zusätzlich die Notwendigkeit von p38 MAPK für wildtypisches Timing der Abendaktivität sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verzögerten Beginn der Abendaktivität und stark verlängerte Freilaufperioden. In Übereinstimmung mit Effekten auf das Laufverhalten scheint darüber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila’s wichtigsten Uhrneuronen eine verspätete nukleäre Translokation von Period zur Folge zu haben. Westernblots legen zusätzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen mögliche Erklärung für den verspäteten Kerneintritt des Uhrproteins. Abschließende Stützung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase“ hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren könnte.
Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die Möglichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.
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.
p21-aktivierte Kinasen regulieren zahlreiche zelluläre Prozesse, die während der Entwicklung, aber auch beispielsweise bei der Krebsentstehung, von zentraler Bedeutung sind. Mbt, das einzige Typ II PAK-Protein von Drosophila melanogaster, spielt eine Rolle bei der Gehirnentwicklung. Eine Nullmutation von mbt, mbtP1, bildet kleinere Gehirne mit stark verkleinerten Pilzkörpern aus. In dieser Arbeit wurde die Funktion von Mbt in Neuroblasten untersucht. Mbt wurde als Teil des apikalen Proteinkomplexes in Neuroblasten des Zentralhirns nachgewiesen. Die apikale Lokalisation von Mbt ist Zellzyklus-abhängig und wird über Bindung an Cdc42 reguliert. Sie ist essentiell für die Funktion von Mbt in Neuroblasten. Trotz apikaler Mbt-Lokalisation in Neuroblasten zeigte die mbt Nullmutante keine Defekte des basalen Mechanismus der asymmetrischen Zellteilung. Mud zeigte geringfügige Lokalisationsveränderungen, die auf einen möglichen Einfluss von Mbt hinweisen. Obwohl PAKs zentrale Regulatoren des Zytoskeletts sind, zeigte die mbtP1 Mutante keine offensichtlichen Veränderungen des Aktin- und Tubulin-Zytoskeletts. Armadillo, ein Aktin-assoziiertes Mbt-Substrat, zeigte ebenfalls keine Lokalisationsveränderung in Neuroblasten. Mbt steuert jedoch die apikale Anreicherung von Cno, einem weiteren Aktin-assoziierten Protein, in Neuroblasten. Darüber hinaus beeinflusst Mbt die Zellgröße von Neuroblasten, sowie deren Proliferationspotenzial und Überleben. mbtP1 Neuroblasten sind kleiner als wildtypische Neuroblasten, haben ein geringeres Proliferationsvermögen und eine geringere Überlebenswahrscheinlichkeit. Der Zelltod von Neuroblasten ist jedoch ein sekundärer Effekt. Daher kann eine Blockierung von Apoptose den adulten Pilzkörperphänotyp nicht retten. Signalwege, die Zellgröße und Proliferation regulieren, wurden auf eine Beteiligung von Mbt hin analysiert. mbtP1 induzierte leichte Effekte im Insulin-Signalweg und die Delokalisation eines nukleolären Proteins. Eine genetische Interaktion von mbtP1 mit Mutationen in Genen des klassischen MAPK-Signalweges identifzierte mbt als Positivregulator dieses Signalweges im Auge. Ein ähnlicher, schwächerer Effekt wurde auch bzgl. der Proliferation und Größe von Neuroblasten beobachtet. Eine 2D-Gelanalyse von Larvengehirnen identifizierte Bic und Hsp83 als mögliche von Mbt regulierte Proteine. Diese Arbeit charakterisiert eine bisher unbekannte Funktion der p21-aktivierten Kinase Mbt in neuronalen Stammzellen und liefert damit Ansatzpunkte für eine detaillierte Aufklärung der Funktionsmechanismen von Typ II PAKs bei der Regulation von Zellproliferation und Überleben
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.