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Babcock, Michael Cameron. "Forward and reverse genetic approaches to studying synaptic transmission in Drosophila melanogaster /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/10289.
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Dulac, Amina. "Identification and functional characterization of the neuronal protein VhaAC45L in Drosophila." Thesis, Université Paris sciences et lettres, 2021. http://www.theses.fr/2021UPSLS035.
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Les V-ATPases sont des complexes protéiques des cellules eucaryotes, hautement conservés, associés à la membrane de nombreux organites vésiculaires ou vacuolaires, dont la fonction est d’assurer un niveau approprié d'acidification. Si le mécanisme général du fonctionnement de cette pompe à protons a été bien étudié, on sait par contre relativement peu de choses sur les propriétés spécifiques de la V-ATPase neuronale. Dans les synapses, ce complexe est essentiel pour acidifier les vésicules synaptiques, permettant ainsi aux transporteurs des neurotransmetteurs de les remplir correctement. Notre équipe a identifié une nouvelle protéine essentielle pour la survie de drosophile, dont la séquence la classe dans la famille des protéines associées aux V-ATPases. Plusieurs bases de données suggèrent que cette protéine, que nous avons nommée Lome, puis VhaAC45L, serait exprimée spécifiquement dans le système nerveux. Nos travaux ont confirmé que Lome est spécifique du système nerveux, et ont en outre révélé que sa présence n'est nécessaire que dans les neurones. Sa localisation cellulaire a montré un enrichissement dans les zones synaptiques chez les mouches adultes et les larves. Nous avons donc concentré la suite de notre étude sur la fonction synaptique de Lome, en utilisant la jonction neuromusculaire de la larve comme modèle. En accord avec l’hypothèse d’un dysfonctionnement de la V-ATPase, les larves ayant un niveau de Lome réduit dans les motoneurones présentaient une augmentation anormale du pH interne des vésicules synaptiques, associée à une diminution de la taille quantique, qui est l'amplitude de la réponse postsynaptique à la libération d'une seule vésicule. En conclusion, nos résultats ont permis d’identifier Lome, alias VhaAC45-Like (VhaAC45L) en référence à son plus proche homologue VhaAC45, comme un régulateur spécifique de la V-ATPase neuronaleThe V-ATPases are highly conserved protein complexes of eukaryotic cells, associated with the membranes of many vesicular or vacuolar organelles, whose function is to ensure an appropriate level of acidification. While the general functioning mechanism of this proton pump has been well studied, in contrast relatively little is known about the specific properties of neuronal V-ATPase. In synapses, this complex is essential to acidify synaptic vesicles, thus allowing neurotransmitter transporters to properly fill them. Our team identified a novel protein essential for Drosophila survival, predicted from its sequence to belong to the family of V-ATPase-associated proteins. According to several databases, this protein, that we named Lome, and then VhaAC45L, appears to be expressed specifically in the nervous system. Our work confirmed that Lome is specific to the nervous system, and further revealed that its presence is only required in neurons. Its cellular localization showed an enrichment in synaptic areas in both adult flies and larvae. We have therefore focused the next part of our study on the synaptic function of Lome, using the larval neuromuscular junction as a model. Consistent with the hypothesis of a V-ATPase dysfunction, larvae with a decreased level of Lome in motoneurons presented an aberrant increase in the internal pH of synaptic vesicles, associated with a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single vesicle. Overall, our results identified Lome, alias VhaAC45Like (VhaAC45L) in reference to its closest homolog VhaAC45, as a specific regulator of the neuronal V-ATPase
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MacNamee, Sarah, and Sarah MacNamee. "Drosophila melanogaster Astrocytes Respond to and Modulate Synaptic Transmission: A Correlative Anatomical and Electrophysiological Study." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621310.
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Astrocytes are the most abundant non-neuronal cells in vertebrate brains. Although Drosophila melanogaster has fewer astrocytic cells relative to neuronal and other glial cell populations, they, like vertebrate astrocytes, are located in synaptic regions, organized into exclusive, minimally-overlapping domains, and play developmental roles in synaptogenesis. But, do Drosophila astrocytes have parallel roles in the regulation of synaptic signaling? Preliminary electron microscopic (EM) data indicates that astrocytic processes are located at a greater distance, on average, from Drosophila synapses than they are from vertebrate synapses, thus raising questions about their capacity to alter synaptic signals. Do astrocytic cells and processes occupy stereotyped synaptic regions across repeating segmental structures and across individuals? In the studies presented here, we have addressed these questions directly in the ventral nerve cord (VNC) of the third-instar larva. We collected the first whole-cell patch-clamp recordings from Drosophila astrocytes. These indicate that intrinsic membrane properties, such as low membrane resistance, high capacitance, a hyperpolarized resting potential relative to neurons, a passive current-voltage relationship, coupling to other astrocytic cells, and an absence of voltage-gated currents, are shared between astrocytes of highly divergent species. Next, we optogenetically activated of a group of glutamatergic pre-motor neurons and showed that astrocytes respond with a glutamate transporter current that is mediated by Eaat1, and that acute, pharmacological and chronic, genetic blockades of this transporter have subsequent effects on the decay of post-synaptic motor neuron currents. Then, we used three-dimensional EM to locate the pre-motor glutamatergic neurons that were activated in the physiological study and measured the distance from each presynaptic site to the nearest astrocytic process. We found that these distances vary 100-fold even along a single neurite and that these structures are rarely in direct contact, but that no synapse is positioned greater than one micron from an astrocytic process. Thus, it is in this anatomical configuration that the regulation of post-synaptic currents by Eaat1 occurs. Finally, we generated a library of single, fluorescently-labeled astrocytes that were co-labeled with fiduciary landmarks, and used this library to compare the placement of astrocyte cell bodies and arbors across VNC segments and individuals. We found substantial variation in the gross shape, size, and territory covered by astrocytes, and conclude that their neuropil domains are not reliably stereotyped. Given the consistent placement of neuronal connectome elements, this indicates that signals of a specific synapse are not regulated by a designated astrocyte. Together, these findings reveal new functional parallels between Drosophila and vertebrate astrocytes. These findings argue for the relevance and applicability of mechanistic discovery in Drosophila astrocytes, and set the stage for further inquiry into the genetic determinants of astrocyte morphology and physiology.
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Muthukumar, Allie. "Astrocyte-Neuron Interactions Regulate Nervous System Assembly and Function: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/745.
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Astrocytes densely infiltrate the brain and intimately associate with synaptic structures. In the past 20 years, they have emerged as critical regulators of both synapse assembly and synapse function. During development, astrocytes modulate the formation of new synapses, and later, control refinement of synaptic connections in response to activity dependent cues. In a mature nervous system, astrocytes modulate synapse function through a variety of mechanisms. These include ion buffering, neurotransmitter uptake and the release of molecules that activate synaptic receptors. Through such roles, astrocytes shape the structure and function of neuronal circuits. However, how astrocytes and synapses reciprocally communicate during circuit assembly remains an unanswered question in the field. The vast majority of our understanding of astrocyte biology has come from studies conducted in mammals, where it is challenging to dissect molecular mechanisms with cell type specificity. Drosophila melanogaster is a less established model system for studying astrocyteneuron interactions, but its vast array of genetic tools and rapid life cycle promises great potential for precisely targeted manipulations. My thesis work has utilized Drosophila melanogaster to investigate the reciprocal nature of astrocyte-synapse communication. First, I characterized Drosophila late metamorphosis as a developmental stage in which astrocyte-synapse associations can be studied. My work demonstrates that during this time, when the adult Drosophila nervous system is being assembled, synapse formation relies on the coordinated infiltration of astrocyte membranes into the neuropil. Next, I show that in a reciprocal manner, neural activity can shape astrocyte biology during this time as well and impart long lasting effects on neuronal circuit function. In particular expression of the astrocyte GABA transporter (GAT) is modulated in an activity-dependent manner via astrocytic GABABR1/2 receptor signaling. Inhibiting astrocytic GABABR1/2 signaling strongly suppresses hyperexcitability in a Drosophila seizure model, vii arguing this pathway is important for modulating excitatory/inhibitory balance in vivo. Finally, utilizing the ease of the Drosophila system, I performed a reverse genetic screen to identify additional astrocyte factors involved in modulating excitatory-inhibitory neuronal balance.
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Muthukumar, Allie. "Astrocyte-Neuron Interactions Regulate Nervous System Assembly and Function: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/745.
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Astrocytes densely infiltrate the brain and intimately associate with synaptic structures. In the past 20 years, they have emerged as critical regulators of both synapse assembly and synapse function. During development, astrocytes modulate the formation of new synapses, and later, control refinement of synaptic connections in response to activity dependent cues. In a mature nervous system, astrocytes modulate synapse function through a variety of mechanisms. These include ion buffering, neurotransmitter uptake and the release of molecules that activate synaptic receptors. Through such roles, astrocytes shape the structure and function of neuronal circuits. However, how astrocytes and synapses reciprocally communicate during circuit assembly remains an unanswered question in the field. The vast majority of our understanding of astrocyte biology has come from studies conducted in mammals, where it is challenging to dissect molecular mechanisms with cell type specificity. Drosophila melanogaster is a less established model system for studying astrocyteneuron interactions, but its vast array of genetic tools and rapid life cycle promises great potential for precisely targeted manipulations. My thesis work has utilized Drosophila melanogaster to investigate the reciprocal nature of astrocyte-synapse communication. First, I characterized Drosophila late metamorphosis as a developmental stage in which astrocyte-synapse associations can be studied. My work demonstrates that during this time, when the adult Drosophila nervous system is being assembled, synapse formation relies on the coordinated infiltration of astrocyte membranes into the neuropil. Next, I show that in a reciprocal manner, neural activity can shape astrocyte biology during this time as well and impart long lasting effects on neuronal circuit function. In particular expression of the astrocyte GABA transporter (GAT) is modulated in an activity-dependent manner via astrocytic GABABR1/2 receptor signaling. Inhibiting astrocytic GABABR1/2 signaling strongly suppresses hyperexcitability in a Drosophila seizure model, vii arguing this pathway is important for modulating excitatory/inhibitory balance in vivo. Finally, utilizing the ease of the Drosophila system, I performed a reverse genetic screen to identify additional astrocyte factors involved in modulating excitatory-inhibitory neuronal balance.
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Lepicard, Simon. "Rôle des protéines associées aux microtubules MAP1/Futsch dans l’organisation et le fonctionnement des synapses à la jonction neuromusculaire de drosophile." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T026.
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Les protéines associées aux microtubules (MAP) de structures, telles que celles appartenant à la famille des MAP1 sont connues pour contrôler la stabilité et la dynamique des microtubules (MTs). Elles sont aussi connues pour interagir avec des protéines post-synaptiques telles que les récepteurs GABAergique ou glutamatergique. Cependant, leur rôle pré-synaptique dans la libération de neurotransmetteurs a été très peu étudié. Dans cette thèse, j'utilise l'avantage du modèle Drosophila melanogaster dans lequel il n'y a qu'un seul homologue des MAP1 des vertébrés, nommé Futsch. J'ai étudié la fonction de Futsch à la jonction neuromusculaire (JNM) de larve, où cette protéine n'est trouvée que dans la partie pré-synaptique. Ici, j'ai montré qu'en plus de sa fonction connue sur la morphologie de la JNM (Roos et al., 2000; Gogel et al., 2006), Futsch est également important pour la physiologie de la JNM, par le contrôle de la libération de neurotransmetteurs ainsi que de la densité des zones actives (ZAs). J'ai montré que l'effet physiologique de Futsch n'est pas la conséquence de l'altération du cytosquelette de MTs ou d'un défaut de transport axonal, mais doit être la conséquence d'un effet local de Futsch à la terminaison synaptique. J'ai utilisé la microscopie d'éclairage structuré 3D (3D-SIM) pour étudier plus précisément la localisation de Futsch et des MTs au niveau de la ZA. Futsch et les MTs se trouvent presque toujours à proximité des ZAs, avec Futsch en position intermédiaire entre les MTs et les ZAs. En utilisant la technique de « proximity ligation assays », j'ai aussi démontré la proximité fonctionnelle de Futsch avec Bruchpilot un composant de la ZA, ce qui n'est pas le cas des MTs. En conclusion, mes données sont en faveur d'un modèle pour lequel Futsch stabilise localement les ZAs, en renforçant leur lien avec le cytosquelette de MTs sous-jacentStructural microtubule associated proteins like those belonging to the MAP1 family are known to control the stability and dynamics of microtubules (MTs). They are also known to interact with postsynaptic proteins like GABA or glutamate receptors. However, their presynaptic role in neurotransmitter release was barely studied. Here, we took advantage of the Drosophila model in which there is only one MAP1 homologue, called Futsch. We studied the function of Futsch at the larval neuromuscular junction (NMJ), where this protein is found presynaptically only. Here, we show that, in addition to its known function on NMJ morphology (Roos et al., 2000; Gogel et al., 2006), Futsch is also important for NMJ physiology, by controlling neurotransmitter release as well as active zone density. We show that this physiological effect of Futsch is not the consequence of disrupted microtubule bundle and disrupted axonal transport, but must be the consequence of a local effect of Futsch at the synaptic terminal. We used 3D-Structured Illumination Microscopy (3D-SIM) to further study the localization of Futsch and MTs with respect to active zones. Both Futsch and MTs are almost systematically present in close proximity active zones, with Futsch being localized in-between MTs and active zones. Using proximity ligation assays, we further demonstrated the functional proximity of Futsch, but not MTs, with the active zone component Bruchpilot. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying MT cytoskeleton
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Schmidt, Manuela. "Characterization of synaptic protein complexes in Drosophila melanogaster." Doctoral thesis, [S.l.] : [s.n.], 2006. http://webdoc.sub.gwdg.de/diss/2006/schmidt.
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Hoeffer, Jr Charles Albert. "Acute activation of conserved synaptic signaling pathways in Drosophila melanogaster." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/280707.
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Studies of memory have identified several memory classifications: declarative, implicit, working, and anesthesia-resistant. One simple classification that may be applied to the array of model systems now used to explore memory is the requirement for de novo gene expression and protein synthesis for the formation of long-term memory (LTM). Short-term memory (STM) appears to require the modification of pre-existing neuronal molecules and is resistant to inhibitors of protein synthesis. These molecules, believed to encode proteins that effect long-lasting neuronal changes likely at the level of the synapse, are manifested behaviorally as memory. Neural activity regulates the cellular decision to synthesize these molecules, yet the identity and function of these molecules are largely unknown. What is known has largely been elucidated by work in mollusks and vertebrates in which procedures have been developed to generate neural activity sufficient to induce long-lasting, protein synthesis-dependent neuronal plasticity. Using these procedures, several key intracellular signaling pathways (Ras/ERK, cAMP/PKA) and important early gene products (arc, zif268, AP1) critical to memory have been identified. Similar procedures are not presently available in Drosophila. Establishing these procedures would greatly enhance the Drosophila model system for identification of plasticity molecules and mechanisms that control their expression. We have explored the potential of conditional Drosophila seizure mutants of comatose and CaP60A mutants for the development of a neural activity generation paradigm capable of (1) inducing long lasting and robust neural activity; (2) acute and persistent activation of the ERK signaling pathway and induction of Drosophila homologs of immediate early genes known to be involved in plasticity; (3) alteration of synaptic localization of fasciclin II, a known effector of synaptic plasticity. Using these mutants, we have established the conservation in insects of a known neural activity regulated signaling pathway shown to be critical to both long term plasticity and memory. Secondly, we have identified a central role for AP1, a classical activity induced gene, in regulating Drosophila neural plasticity. The neural activity paradigm coupled with the identification AP1 dual control of both major branches of long term neuronal change, structural and functional plasticity, provides researchers valuable tools for addressing some the outstanding questions facing the plasticity field today.
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Bucher, Daniel. "An Electrophysiological Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction." Doctoral thesis, kostenfrei, 2008. http://www.opus-bayern.de/uni-wuerzburg/volltexte/2008/2778/.
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Lockyer, Rebecca. "Transmission of chemosensory information in Drosophila melanogaster : behavioural modification and evolution." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/transmission-of-chemosensory-information-in-drosophila-melanogaster-behavioural-modification-and-evolution(45101800-3913-4a6b-8c46-52153cbcc3f3).html.
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The ‘modern evolutionary synthesis’ emphasised the role of genetic inheritance in driving natural selection; however, this is not the only means by which biological changes may be passed on to future generations. Information can also be transmitted non-genetically, and this could be an important agent of evolution. Non-genetic information can be acquired in two different ways: it can be inherited from parents (for example, through maternal and paternal effects) or gathered from the environment. Transmission of information in this manner can result in durable changes in behaviour, which allow for adaptation to variable conditions, and might ultimately bring about adaptive divergence. To investigate non-genetic transmission of information between parents and offspring, I studied the effects of being reared in the presence of an aversive stimulus, peppermint extract, on the fruit fly Drosophila melanogaster using a range of behavioural assays. The results demonstrate that naïve flies exposed to peppermint found it aversive, with exposure substantially reducing their survival; however, the offspring of flies reared in the presence of peppermint showed a significantly reduced aversion despite having no previous direct contact with the stimulus. This strongly suggests that a transmission of information (relating to preference for peppermint) has occurred from parents to offspring. This effect was preserved for four generations if the peppermint stimulus was removed from the food source after only one generation, but with continued exposure to peppermint the reduction in aversion was sustained, and a preference for peppermint may even have developed. Mutant flies lacking OrCo, Trp and Painless showed abnormal behavioural responses to peppermint, suggesting that these genes may be involved in detecting and/or responding to this aversive stimulus. These experiments demonstrate that environmental changes (i.e. the introduction of an aversive stimulus) can instigate biological modifications in D. melanogaster that are passed on non-genetically to future generations. This is most likely true for other insects and animals more generally, and further studies of additional model and non-model species will help to demonstrate the importance and prevalence of non-genetic transmission of information as a driver of fundamental evolutionary change.
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Li, Yihang. "Mechanisms of Synaptic Development and Premature Aging in Drosophila: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/862.
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Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction.
In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.
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Yuan, Ning. "DISTINCT MODULATORY EFFECTS OF DOPAMINE ON EXCITATORY CHOLINERGIC AND INHIBITORY GABAERGIC SYNAPTIC TRANSMISSION IN DROSOPHILA." Ohio University / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1149001533.
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Li, Jingjun Bhat Manzoor A. "Crucial role of Drosophila neurexin in proper active zone apposition to postsynaptic densities, synaptic growth and synaptic transmission." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1919.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Neurobiology, School of Medicine." Discipline: Neurobiology; Department/School: Medicine.
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Xing, Xiaomin. "Genetic and functional analysis of synaptic CA²⁺ dynamics in Drosophila." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/2168.
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Ca²⁺ influx is one of the critical events that trigger synaptic vesicular release, and the accumulation of residual free Ca²⁺ in synapses is also important for activity-dependent synaptic plasticity. Ca²⁺ imaging with fluorescence indicators (synthetic or genetically encoded) is a powerful approach to monitor Ca²⁺ levels in neurons and synapses. Although accumulating studies in vertebrate systems have been carried out to demonstrate the role of Ca²⁺ in synaptic transmission and plasticity, most of these studies rely on pharmacological methods to infer the molecular mechanism, with less emphasis on forward genetic analysis. The Drosophila neuromuscular junction (NMJ) is a powerful neurogenetic platform for studying synaptic transmission, because of the availability of many mutations. However, not many mutations have been analyzed with Ca²⁺ imaging. Besides, although Genetically Encoded Ca²⁺ Indicators (GECIs) including GCaMPs are increasingly popular as the tool to identify neuronal circuits activated by certain stimuli or mediating particular behaviors, the physiological and functional interpretation of neuronal Ca²⁺ transients reported by GECIs remain obscure.
By expressing GCaMPs in NMJ synapses, I characterized a spectrum of genetic mutations including sodium channel alleles parats¹, parabss¹, potassium channel mutations Shaker (ShM, Sh¹²⁰), Shab³, ether-a-go-go (eag¹, eag⁴pm), and double mutant eag¹ Sh¹²⁰. Drosophila NMJs contain at least three different types of synapses, which include glutamatergic tonic motor synapse type Ib, phasic motor synapse type Is, and modulatory octopaminergic synapse type II. In this study, I found that the ion channel mutations did not uniformly alter the Ca²⁺ dynamics in type Ib, Is and II synapses. Based on genetic dissection and pharmacological analyses, I concluded that the excitability type I and type II synapses are differentially regulated by various ion channels, and that ion channels mainly influence the influx of Ca²⁺ upon membrane depolarization but not the subsequent clearance.
I also attempted to interpret the significance of synaptic Ca²⁺ transients by correlating Ca²⁺ imaging with electrophysiological recordings. One important gap in the application of GCaMP indicators is its postsynaptic physiological relevance. Correlation of synaptic GCaMP Ca²⁺ transients with postsynaptic currents simultaneously recorded by focal extracellular recording indicated that Ca²⁺ transients reported by GCaMPs were slow, and did not reflect immediate synaptic transmission. Rather, the kinetics of synaptic Ca²⁺ transients was temporally correlated with short-term synaptic plasticity such as facilitation and depression. The hyperexcitable ion channel mutations Sh and parabss¹ enhanced the synaptic Ca²⁺ transient amplitudes as well as depression. Type Is synapses of hyperexcitable mutations such as eag¹ Sh¹²⁰ and parabss¹ often displayed single stimulus pulse-evoked Ca²⁺ transients, which were induced by high frequency repetitive firing of action potentials. Such Ca²⁺ transients were correlated with supernumerary peaks of postsynaptic currents. Based on the slow kinetics and the correlation with short-term plasticity, I conclude that GCaMP Ca²⁺ signals better reflect the accumulation of cytosolic residual Ca²⁺. The spontaneous Ca²⁺ waves in larval motor neurons were well correlated with high frequency nerve action potentials, suggesting that accumulation of residual Ca²⁺ occurs in larval crawling.
Overall, this study provided important information about the different excitability control and Ca²⁺ clearance mechanisms in different synapses, and examined how membrane excitability controls the influx and accumulation of synaptic cytosolic residual Ca2+, as indicated by GCaMPs. Further, by correlating synaptic Ca²⁺ dynamics with electrophysiology, this study also investigated how to interpret GCaMP Ca²⁺ signals in the context of facilitation and depression, establishing a basis for an integrated approach of studying short-term synaptic plasticity from complementary physiological signals.
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Tian, Rui [Verfasser], and Stephan [Akademischer Betreuer] Sigrist. "Structural and functional organization of synaptic proteins in Drosophila melanogaster / Rui Tian. Betreuer: Stephan Sigrist." Würzburg : Universitätsbibliothek der Universität Würzburg, 2011. http://d-nb.info/1013619986/34.
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Mende, Michael. "Analysing the role of short stop during the formation of synaptic terminals in Drosophila melanogaster." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971998396.
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Shayan, Alexander Joseph. "The role of the drosophila learning and memory gene dunce (DNC) in synaptic transmission." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0005/MQ40876.pdf.
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Dagaeff, Anne-Cécile. "Selection, sex and sun : social transmission of a sexual preference in Drosophila melanogaster." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30208/document.
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Choisir un partenaire est une décision importante pour les organismes à reproduction sexuée. Une possibilité est de copier le choix d'autres individus, c'est ce que l'on appelle l'imitation du choix du partenaire, phénomène bien étudié chez les vertébrés, mais peu connu chez les invertébrés. Cette thèse porte sur l'imitation du choix du partenaire chez la drosophile (Drosophila melanogaster). J'ai tout d'abord montré que les femelles pouvaient être influencées dans leur choix par l'observation d'une seule autre femelle et que ceci était corrélé avec la pression atmosphérique. J'ai ensuite étudié si la préférence pour un type de mâle pouvait être transmise au sein de la population. Enfin j'ai commencé un travail exploratoire pour identifier les molécules impliquées dans ce processus d'imitation. Ces résultats montrent que les drosophiles peuvent exprimer des comportements complexes conduisant potentiellement à la transmission culturelle, l'isolement reproductif et la spéciationMate choice is a major fitness-affecting decision in sexually reproducing organisms. A form of mate choice is mate copying, in which females choose potential mates by copying the mate choice of conspecifics. While many studies documented mate copying in vertebrates, little is known about this behaviour in invertebrates. In this thesis, I studied mate copying in Drosophila melanogaster females. I showed that female flies can build a sexual preference for one male characteristic after witnessing a single mate choice event and that the efficiency of mate copying correlates with air pressure and its variations. Then I studied the characteristics of mate copying to see whether a preference for one type of male can be transmitted into the population. Finally I tried to find some molecules that could be involved in this behaviour. These results indicate that fruit flies can express complex behaviour, which can potentially lead to cultural transmission, reproductive isolation and speciation
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Debec, Alain. "Etude génétique de lignées cellulaires de Drosophila melanogaster." Paris 6, 1986. http://www.theses.fr/1986PA066180.
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Etude de 25 systèmes isoenzymatiques dans 18 lignées de cellules embryonnaires de d. M. ; 7 lignées permanents ont été établies in vitro à partir d'embryons létaux haploïdes. A partir d'un clone haploïde, une lignée cellulaire résistante au cadmium a été sélectionnée. Ces cellules présentent un niveau de métallothionéine 22 fois plus élevé que le clone d'origine. Clonage du cdna d'une métallothionéines. Une lignée haploïde présente des anomalies mitotiques (absence de centriole observée en microscopie électronique).
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Li, Yihang. "Mechanisms of Synaptic Development and Premature Aging in Drosophila: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/862.
Full textAbstract:
Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction.
In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.
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Stanback, Alexandra Elizabeth. "The Effects of a Ketone Body on Synaptic Transmission." UKnowledge, 2019. https://uknowledge.uky.edu/biology_etds/57.
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The ketogenic diet is commonly used to control epilepsy, especially in cases when medications cannot. The diet typically consists of high fat, low carb, and adequate protein and produces a metabolite called acetoacetate. Seizure activity is characterized by glutamate excitotoxicity and therefore glutamate regulation is a point of research for control of these disorders. Acetoacetate is heavily implicated as the primary molecule responsible for decreasing glutamate in the synapse; it is believed that acetoacetate interferes with the transport of glutamate into the synaptic vesicles. The effects on synaptic transmission at glutamatergic synapses was studied in relation to the ketogenic diet in Drosophila larvae for this thesis. Various measures of synaptic transmission were conducted. Acetoacetate decreased neurotransmission at the synapse. It was also found that acetoacetate has direct effects on the postsynaptic membrane, which indicates a novel role for the metabolite.
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Panchumarthi, Sarvari. "The Drosophila Serrate is Required for Synaptic Structure and Function at Larval Neuromuscular Junctions." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/194269.
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Drosophila melanogaster is an excellent model system to identify genes involved in synaptic growth and function. In Drosophila, the Serrate (Ser) gene encodes a transmembrane protein that is a ligand for Notch receptor. Several previous studies implicated a role for Serrate in normal wing development and patterning. In this study, I demonstrate that Serrate is required for normal synaptic growth and function. I characterized the phenotype of a Serrate mutation (serB936) that was identified by an EMS-induced genetic screen aimed at identifying novel genes that play a role in synaptic growth and function. Co-localization studies show that Serrate protein is expressed at both the pre- and postsynaptic side of larval neuromuscular junctions (NMJs). Mutations in ser impair synaptic transmission at larval NMJs. This defect is entirely presynaptic, as nerve-evoked excitatory junction potentials (EJP) and quantal content (QC) of neurotransmitter release are significantly reduced when compared to wild-type control. Further, mutations in ser also alter the growth of the NMJ and the underlying muscle. Mutations in ser significantly reduce the size of larval body wall muscles (length and surface area) as well as the number and size of synaptic boutons, and the number of secondary axonal branches. Ubiquitous or muscle-specific expression of normal Serrate in serB936 mutants restores a normal muscle size but not a normal size and structure of the innervating NMJ. However, expression of normal Serrate in the motor axon restores a normal number of synaptic boutons and secondary branches at serB936 mutant NMJs. In addition, it restores normal neurotransmitter release. These data suggest that Serrate protein is required presynaptically for normal synaptic growth and function. Interestingly, overexpression of Serrate in a wild type background resulted in similar phenotypes than to those of loss-of-function mutants. In conclusion, these data suggest a new functional role for Serrate in synaptic growth and function.
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Habayeb, Mazen. "Nora virus as a model to study persistent infection in Drosophila melanogaster." Doctoral thesis, Molecular Biology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-22129.
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Drosophila melanogaster has been widely used as a model organism to study the immune responses against bacteria, fungi, parasites and viruses. Here, I present a D. melanogaster virus as a model to study persistent virus infections. I have discovered and characterized the Nora virus, a small picorna-like RNA virus able to persistently infect D. melanogaster. The Nora virus genome encodes four open reading frames; a feature not present in other picorna-like viruses. The Nora virus is not closely related to any other virus family, but rather is the first virus in a new family of picorna-like viruses. The major replicative proteins of this virus are encoded in the second open reading frame and the capsid proteins are encoded in the fourth open reading frame. The sequence of the capsid proteins are not obviously related to any other previously described protein. By looking at expressed sequence tags (EST) projects, we identified an EST sequence from the parasitic wasp Nasonia which appears to encode proteins that have sequence similarity to the Nora virus capsid proteins. I have shown that the Nora virus persists in the fly intestine however I did not observe serious pathological effects in the infected flies. The virus is shed through feces and the transmission occurs horizontally via the ingestion of virus-contaminated food. Moreover, I observed variability in the viral titers among single flies of the same infected stock. Some flies are able to clear the Nora virus but not others and this phenomenon seems to be titer-dependent. Surprisingly, none of the known Drosophila antiviral responses play a role against the Nora virus. In conclusion, my work shows that studying the Nora virus interaction with the Drosophila immune system can lead to new findings on viral persistence mechanisms of RNA viruses and of Drosophila viral innate immunity.
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Touret, Franck. "Etude in vivo et in vitro du rétrovirus endogène gypsy chez Drosophila melanogaster." Paris, EPHE, 2014. http://www.theses.fr/2014EPHE3001.
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Les rétrovirus endogènes sont le résultat d’infections successives des cellules germinales de leurs hôtes par des rétrovirus. Cette infection avec transmission mendélienne du génome rétroviral est nommée endogénisation et correspond à un processus encore peu connu Drosophila melanogaster constitue un modèle de choix pour comprendre ce processus grâce à la présence naturelle du rétrovirius endogène gypsy. Chez les individus permissifs ce rétrovirus est d’abord exprimé au niveau d’une couche de cellules somatiques, puis il est ensuite transféré dans l’oovocyte. Chez les individus restrictifs, cette expression est alors réduite par les piRNAs, générés par le locus flamenco et ciblant les ARNs transcrits de gypsy. Dans ce travail, nous avons démontré que la bactérie endosymbiotique Wolbachia était capable de réduire la transmission maternelle lorsqu’elle était présente en même temps que le rétrovirus gypsy dans l’ovocyte, et ce , indépendamment des piRNAs Wolbachia représente donc le facteur négatif limitant l’invasion du généome de l’hôte par gypsy. Nous avons aussi isolé, à partir d’une culture de cellules embryonnaires, une forme infectieuse de gypsy capable de former des partcicules virales indectieuses et d’oinfecter une autre lignée de cellules. Les mécanismes permettant de rester infectieux sont encore inconnus, mais ces résultats pourraient aider à mieux comprendre le processus générald’endogénisation des retrovirus. L’ensemble de ce travail décrit ainsi plus précisemment les relations intimes et l’équilibre entre un hôte, ses habitants et un rétrovirusendogèneRetroviruses are viruses that have the ability to reverse transcribe their RNA genome into DNA to integrate it in the host genome. Sequencing of many eukaryotic genomes has revealed the presence of many of these endogenous retrovirus sequences. The mechanisms by which these retroelement colonize the genome are still unknown. The endogenous retrovirus gyspsy of Drosophilz melanogaster is a powerful experimental model to decipher this process. Gypsy is expressed in a sheet of somatic cells and transformed into the oocyte. This critical step is the first of the endogenization process which remains poorly understood. In this work we have shown that gypsy maternal transmission is reduced in the presence of the endosymbiotic bacteria Wolachia in a piRNAs independent way. Wolbachia represent a negative factor limiting gypsy genome invasion. We also isolate from a cell culture an infectious form of the gypsy endogenenous retrovirus. This form of gypsy is still able to form enveloped viral particles ans still have infectious properties. This process by which this virus can bud from the cell and still be infectious is unknown but i can lead to a better understanding of the endogenization process. Together this findings allow a better understanding of the complex relationship between host (and his inhabitants) and an endogenous retrovirus
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Grentzinger, Thomas. "Caractérisation moléculaire de la transmission épigénétique d’un caractère acquis, la régulation de l’élément I chez Drosophila melanogaster." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20038/document.
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Les cellules, et tout particulièrement les cellules germinales, maintiennent l'intégrité de leur génome en prévenant d'éventuelles mutations comme celles dues à la mobilité des éléments transposables (ET). Dans la lignée germinale des animaux, une classe particulière de petits ARN régulateurs, les PIWI-interacting RNA (piRNA), sont les acteurs majeurs du contrôle des ET. Chez Drosophila melanogaster, il existe des souches dites réactives, dépourvues de copies actives de l'élément I, ET exprimé dans la lignée germinale femelle. Les femelles de ces souches voient leur capacité à réprimer l'invasion de leur génome par l'élément I augmenter avec l'âge. Des données antérieures ont montré qu'une fois acquise, la capacité à réprimer l'élément I est transmise maternellement au travers des générations. Mes travaux de thèse ont permis de montrer que la transmission de la capacité à réprimer l'élément I n'est pas corrélée à des modifications de l'activité transcriptionnelle des loci producteurs de piRNA, mais semble uniquement véhiculée par les piRNA. En effet, les piRNA de l'élément I déposés dans l'embryon vont amorcer la production de piRNA complémentaires dans les ovaires de la descendance, ce qui induit une forte accumulation de piRNA antisens à l'élément I. Ainsi, les piRNA maternellement déposés assurent la transmission de la capacité à réprimer l'élément I, acquise suite au vieillissement des ascendants maternels. Mes résultats mettent en évidence le rôle des piRNA comme support moléculaire d'une composante non génétique de l'information héritable, indépendante de la chromatine et déterminante pour le maintien de l'intégrité du génomeCells, especially germinal stem cells, maintain genomic integrity by averting the propagation of mutations, generated as a consequence of DNA damage. In particular, they must avoid the deleterious activity of transposable elements (TEs). In animal germlines, one of the key players of the TE repression involves a specific class of small regulatory RNAs, the PIWI-interacting RNAs (piRNAs). In Drosophila melanogaster, there are reactive strains that are devoid of functional copies of the I element, a TE specifically expressed in the female germ line. When they get older, females of these strains can acquire a strong capacity to repress the I element invasion. Anterior works have shown that once acquired, this capacity to repress the I element is maternally transmitted over generations. The results obtained during my thesis revealed that the transmission of the capacity to repress the I element is not correlated with increased transcriptional activity of piRNA producer loci but seems only mediated by the piRNAs. Indeed, I element piRNAs deposition in the embryo after aging treatment correlates with the production of complementary piRNAs in the ovaries of the progeny. This results in a strong accumulation of antisense I element piRNAs. The maternally deposited piRNAs ensure the transmission of the capacity to repress the I element acquired after ancestor aging. My results highlight the molecular support of a DNA- and chromatin-independant component of heritable information essential for the maintenance of genome integrity
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Engelhardt, Frauke [Verfasser], and Stephan [Gutachter] Sigrist. "Synaptic Connectivity in the Mushroom Body Calyx of Drosophila melanogaster / Frauke Engelhardt [geb. Christiansen]. NeuroCure Charité Berlin. Gutachter: Stephan Sigrist." Würzburg : Universität Würzburg, 2013. http://d-nb.info/1112089853/34.
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Bilz, Florian [Verfasser], André [Akademischer Betreuer] Fiala, André [Gutachter] Fiala, and Ralf [Gutachter] Heinrich. "Optical Analysis of Synaptic Plasticity Underlying Associative Learning in Drosophila melanogaster / Florian Bilz ; Gutachter: André Fiala, Ralf Heinrich ; Betreuer: André Fiala." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2018. http://d-nb.info/1195215843/34.
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Zhu, Yuechen. "THE EFFECT OF COLD ON THE PHYSIOLOGY OF DROSOPHILA LARVA HEART AND ON SYNAPTIC TRANSMISSION AT CRAYFISH NEUROMUSCULAR JUNCTIONS." UKnowledge, 2017. http://uknowledge.uky.edu/biology_etds/50.
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Ectothermic animals are susceptible to temperature changes such as cold shock with seasons. To survive through a cold shock, ectotherms have developed unique strategies. My interest is focusing on the physiological function of during cold shock and prolonged cold exposure in the fruit fly (Drosophila melanogaster) and crayfish (Procambarus clarkii). I used Drosophila melanogaster as a model system to investigate cardiac function in response to modulators (serotonin, acetylcholine, octopamine, dopamine and a cocktail of modulators) in acute cold shock and chronic cold shock conditions as possible mechanism to regulate heart rate in the cold. To examine if the dampened heart rate in the cold could still be enhanced by modulators or calcium loading, modulators and light-sensitive channelrhodopsin proteins were utilized to stimulate the heart. This light induced cardiac activation increased heart rate in all conditions, and potentially can be used for cardiac therapy in mammals. Also, the acute and chronic cold conditioned heart showed responsiveness to the above mention modulators. In examining how synaptic transmission is influenced by acute and chronic cold, the crayfish neuromuscular junction was used as a model. This is a good model as there are high and low output synapses to be investigated. The low output neuromuscular junction was enhanced in response to acute cold. The high output nmj increased in synaptic response to acute cold. In addressing chronic cold conditions, the nmj were physiologically assayed in their response to acute warm changes as well as influence of serotonin and octopamine. In chronic cold condition, the synaptic output was varied in enhanced and dampened responses to an acute warm environment. These junctions were enhanced in their synaptic output by serotonin and octopamine (100nM). In assessing, by HPLC assay, octopamine concentration increased in chronic cold crayfish. This suggests compensation in synaptic transmission in cold acclimation possibility via endocrine responses.
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Fuentes, Medel Yuly F. "Role of Glia in Sculpting Synaptic Connections at the Drosophila Neuromuscular Junction: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/580.
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Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ).
During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth.
My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis.
My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.
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Warren-Paquin, Maude. "Regulation of synaptic plasticity at the Drosophila larval NMJ : the role of the small GTPase Rac." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112319.
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We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.
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PERRIN-WALDEMER, CLAUDE GILBERT. "Etude des glandes accessoires du male de drosophila melanogaster (meigen) : cytophysiologie et cytochimie." Clermont-Ferrand 2, 1987. http://www.theses.fr/1987CLF2E359.
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Ganguly, Archan. "The Role of Serotonin-cAMP Mediated Signaling in Drosophila Central Synaptic Transmission and its Implications in Larval Olfactory Associative Learning." Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1331263683.
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Halder, Partho [Verfasser], and Erich [Akademischer Betreuer] Buchner. "Identification and characterization of synaptic proteins of Drosophila melanogaster using monoclonal antibodies of the Wuerzburg Hybridoma Library = Identifikation und Charakterisierung von synaptischen Proteinen von Drosophila melanogaster mit Hilfe von monoklonalen Antikörpern der Würzburger Hybridoma-Bibliothek / Partho Halder. Betreuer: Erich Buchner." Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/101948733X/34.
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Lavige, Jean-Marc. "Systeme i-r de dysgenesie hybride chez drosophila melanogaster : etude du developpement abortif des embryons de meres sf." Clermont-Ferrand 2, 1987. http://www.theses.fr/1987CLF21080.
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Cadieu, Jean-Claude. "Influence de l'expérience individuelle dans la formation de la préférence alimentaire et sa transmission entre les générations : étude chez Drosophila melanogaster et Serinus canarius." Toulouse 3, 2003. http://www.theses.fr/2003TOU30107.
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Rodriguez, Ralph. "Etude de la régulation de la voie de signalisation Hedgehog chez Drosophila melanogaster : diffusion et transmission du signal, caractérisaion de RipCurl, un nouveau régulateur." Nice, 2005. http://www.theses.fr/2005NICE4041.
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The secreted molecules of the family Hedgehog (Hh) play a crucial part during the development at the vertebrate ones and the invertebrates. The way of Hh indication is implied in the appearance of many cancers at the man (cancers of the skin, brain of the prostate and digestive system). From the functional conservation of indication Hh enters the vertebrate ones and the drosophila, the latter remains a model privileged for the functional study of this way. During my thesis, I stuck to two aspects of Hh indication : The control of the diffusion and the movement of Hh since its producing cells, mechanisms of the transduction and the regulation of the Hh signalling the receiving cells. For that I studied the role of the lipidic modifications of Hh and their need in the activation of various target genes of the way. Hh is synthesized in the form of a precursor whose maturation is carried out before its secretion. The secreted factor corresponds to the Nfinal area of the precursor decorated with a cholesterol molecule out of C-terminal and with a palmitic molecule of acid out of N-terminal. I thus could show that the presence of cholesterol on Hh was necessary to its secretion and its diffusion beyond the cells which produce it, and that the presence of the palmitic acid was necessary for the potentiation of the Hh signal. The second part of my thesis is centered on the comprehension of the mechanisms of regulation of the Hh way and its various components. The Hh way consists of a complex of reception of the signal made up of the Patched receiver and of its Co-receiver smoothened (Smo), and of a complex of transduction of the signal whose principal components are: the kinésine costal-2 (Cos), kinase Fused (Fu), the factor of transcription ulna interruptus (Ci). I took part in the demonstration that the transmission of the Hh signal between its complex of reception and the complex of transduction of the signal are done by direct interaction between the co-receptor Smo and Cos. Thereafter, I was interested in the regulation of the stability of the proteins Smo and Cos. For that I carried out a clonal analysis in the disc imaginal of wing for various genes coding for kinases (such as PKA, GSK3) and of proteins of the complexes of ubiquitination (such ascertain components of complexes SCF). All these proteins are known to play a part in the stability and the regulation of the factor of transcription of the Hh way: Ci. The analysis of the loss of function clones for these various genes enabled me to select candidates implied in the stability of Cos. In order to better understand the regulation of the Hh way, I was interested in the characterization of a new gene (ripcurl: RPC) identified at the time of a mutagenesis of deregulation. The over-expression of RPC makes it possible to obtain similar phenotypes with losses of function of hh in the wing. I showed that RPC intervenes between the complex of reception and the cytoplasmic complex of transduction of the Hh signal. I also generated and characterized a mutant of RPC obtained by Re-mobilization of an element P in order to make a phenotypical study. Complementarily, I developed biochemical tools to allow myself to identify his partnersLes molécules sécrétées de la famille Hedgehog (Hh) jouent un rôle crucial au cours du développement chez les vertébrés et les invertébrés. La voie de signalisation Hh est impliquée dans l'apparition de nombreux cancers chez l'homme (cancers de la peau, du cerveau de la prostate et de l'appareil digestif). De par la conservation fonctionnelle de la signalisation Hh entre les vertébrés et la drosophile, cette dernière reste un modèle privilégié pour l'étude fonctionnelle de cette voie. Au cours de ma thèse, je me suis attaché à deux aspects de la signalisation Hh: le contrôle de la diffusion et du mouvement d'Hh depuis ses cellules productrices, es mécanismes de la transduction et de la régulation du signal Hh dans les cellules réceptrices. Pour cela j'ai étudié le rôle des modifications lipidiques de Hh et leur nécessité dans l'activation des différents gènes cibles de la voie. Hh est synthétisé sous forme d'un précurseur dont la maturation est réalisée avant sa sécrétion. Le facteur sécrété correspond à la région N-terminale du précurseur décorée d'une molécule de cholestérol en C-terminal et d'une molécule d'acide palmitique en N-terminal. J'ai ainsi pu montrer que la présence du cholestérol sur Hh était nécessaire à sa sécrétion et à sa diffusion au delà des cellules qui le produisent, et que la présence de l'acide palmitique était nécessaire pour la potentialisation du signal Hh. La deuxième partie de ma thèse s'axe sur la compréhension des mécanismes de régulation de la voie Hh et de ses différents composants. La voie Hh est constituée d'un complexe de réception du signal composé du récepteur Patched et de son co-récepteur smoothened (Smo), et d'un complexe de transduction du signal dont les principaux composants sont : la kinésine costal-2 (Cos), la kinase Fused (Fu), le facteur de transcription cubitus interruptus (Ci). J'ai participé à la démonstration que la transmission du signal Hh entre son complexe de réception et le complexe de transduction du signal se fait par interaction directe entre le co-récepteur Smo et Cos. Par la suite, je me suis intéressé à la régulation de la stabilité des protéines Smo et Cos. Pour cela j'ai effectué une analyse clonale dans le disque imaginal d'aile pour différents gènes codant pour des kinases (tel que PKA, GSK3) et des protéines des complexes d'ubiquitination (tel que certains composants des complexes SCF). Toutes ces protéines sont connues pour jouer un rôle dans la stabilité et la régulation du facteur de transcription de la voie Hh : Ci. L'analyse des clones perte de fonction pour ces différents gènes m'a permis de sélectionner des candidats impliqués dans la stabilité de Cos. Afin de mieux comprendre la régulation de la voie Hh, je me suis intéressé à la caractérisation d'un nouveau gène (ripcurl : rpc) identifié lors d'une mutagenèse de dérégulation. La surexpression de Rpc permet d'obtenir des phénotypes similaires à des pertes de fonction d'hh dans l'aile. J'ai montré que Rpc intervient entre le complexe de réception et le complexe cytoplasmique de transduction du signal Hh. J'ai aussi généré et caractérisé un mutant de Rpc obtenu par re-mobilisation d'un élément P afin de réaliser une étude phénotypique. Complémentairement, j'ai développé des outils biochimiques pour me permettre d'identifier ses partenaires
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Gauthier, Emmanuel. "Le facteur I et la dysgénésie des hybrides chez Drosophila melanogaster : régulation artificielle par des ARN antisens et mise en évidence de phénomènes épigénétiques à transmission bi-parentale." Lyon 1, 1999. http://www.theses.fr/1999LYO10145.
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Le facteur i (fi) est un element transposable de type line de drosophila melanogaster. Les souches dites reactives (r) sont depourvues de cet element a l'etat fonctionnel, les souches dites inductrices (i) en possedent au moins une copie. Le fi obeit a une regulation zygotique et maternelle. Il est stable en contexte inducteur, mais sa mobilisation peut etre induite a haute frequence dans la lignee germinale des femelles issues du croisement entre une femelle r et un male i. Ces femelles dites sf (sterilite femelle), sont fortement steriles suite a une mort precoce des embryons qu'elles pondent. Le croisement reciproque femelle i par male r donne des femelles rsf (reciproque sterilite femelle) qui sont normalement fertiles bien que le fi transpose aussi dans leur lignee germinale. Les caracteristiques de ce phenomene, qui ne touchent pas les descendants males, definissent la dysgenesie des hybrides de type i-r. Le fi transpose via un arn qui sert d'intermediaire de transposition et d'arnm. Afin d'evaluer la reelle implication de la mobilisation du fi dans l'apparition du phenotype de sterilite, nous avons cible ce transcrit en produisant des arn antisens ou sens transgeniques. Toutes nos constructions ciblant la partie 5 du fi permettent de reduire l'expression d'une fusion fi-lacz et la sterilite sf. La transposition est donc responsable de la letalite embryonnaire. Pour un transgene donne, l'efficacite de la repression depend du nombre de copies, de leur mode de transmission, de l'age des femelles mais aussi des lignees, et peut persister dans certains cas plusieurs generations apres elimination du transgene. A l'effet zygotique s'ajoutent des effets maternels, et paternels. A l'oppose, la construction ne ciblant pas la partie 5 augmente, maintient, ou provoque une sterilite dans des contextes normalement peu ou pas steriles. Cet effet activateur est strictement zygotique, et montre que le fi est capable d'autoactivation en trans. La memoire repressive est observee pour des constructions ne produisant pas toutes des arn antisens et/ou sens, et la construction qui ne cible pas la region 5 du fi n'entraine pas de tels effets. Nous proposons donc que l'inhibition observee soit due a la seule presence euchromatique de sequences homologues a la sequence 5 du fi, independamment d'une activite transcriptionnelle. Cette repression de sequences homologues, qui rappelle par certains aspects les phenomenes de co-suppression deja decrits chez les plantes et quelques animaux, est l'evidence de l'implication de mecanismes epigenetiques dans la regulation du facteur i.
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Koon, Alex C. "Autoregulatory and Paracrine Control of Synaptic and Behavioral Plasticity by Dual Modes of Octopaminergic Signaling: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/572.
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Synaptic plasticity—the ability of a synapse to change—is fundamental to basic brain function and behavioral adaptation. Studying the mechanisms of synaptic plasticity benefits our understanding of the formation of neuronal connections and circuitry, which has great implications in the field of learning and memory and the studies of numerous human diseases.
The Drosophila larval neuromuscular junction (NMJ) system is a powerful system for studying synaptic plasticity. The NMJ consists of at least two different types of motorneurons innervating the body wall muscles. Type I motorneurons controls muscle contraction using glutamate as the neurotransmitter, while type II are modulatory neurons that contain octopamine. Octopamine is a potent modulator of behavior in invertebrates. Nevertheless, its function at the synapse is poorly understood.
In my thesis research, I investigated the role of octopamine in synaptic plasticity using the Drosophila NMJ system. Preliminary observations indicate that increased larval locomotion during starvation results in an increase of filopodia-like structures at type II terminals. These structures, which we termed as “synaptopods” in our previous studies, contain synaptic proteins and can mature into type II synapses. I demonstrated that this outgrowth of type II terminals is dependent on activity and octopamine. Mutations and genetic manipulations affecting the production of octopamine decrease synaptopods, whereas increase of type II activity or exogenous application of octopamine increase synaptopods. Interestingly, I found that the type II octopaminergic neurons have an absolute dependence on activity for their innervation of the muscles. Blocking activity in these neurons throughout development results in no type II synapses at the NMJ, whereas blocking activity after the formation of synapses results in gradual degradation of type II terminals.
Next, I examined the autoregulatory mechanism underlying the octopamine-induced synaptic growth in octopaminergic neurons. I discovered that this positive-feedback mechanism depends on an octopamine autoreceptor, Octß2R. This receptor in turn activates a cAMP- and CREB-dependent pathway that is required in the octopamine-induction of synaptopods. Furthermore, I demonstrated that this octopaminergic autoregulatory mechanism is necessary for the larva to properly increase its locomotor activity during starvation.
Thirdly, I investigated the possibility that type II innervation might regulate type I synaptic growth through octopamine. We found that ablation, blocking of type II activity, or the absence of octopamine results in reduced type I outgrowth, and this paracrine signaling is mediated by Octß2R which is also present in type I motorneurons.
Lastly, the function of another octopamine receptor, Octß1R, was examined. In contrast to Octß2R, Octß1R is inhibitory to synaptic growth. I demonstrated that the inhibitory effect of this receptor is likely accomplished through the inhibitory G-protein Goα. Similar to Octß2R, Octß1R also regulates the synaptic growth of both type I and type II motorneurons in a cell-autonomous manner. The inhibitory function of this receptor potentially breaks the positive feedback loop mediated by Octß2R, allowing the animal to reset its neurons when the environment is favorable.
In summary, the research presented in this thesis has unraveled both autoregulatory and paracrine mechanisms in which octopamine modulates synaptic and behavior plasticity through excitatory and inhibitory receptors.
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Tian, Rui. "Structural and functional organization of synaptic proteins in Drosophila melanogaster." Doctoral thesis, 2011. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-57399.
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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 phenotypeEs wird angenommen, dass strukturelle und funktionale Änderungen an synaptischen Verbindungen („synaptische Plastizität”) die Grundlage für Lern- und Gedächtnisprozesse darstellen. Daher sind die molekularen Mechanismen des strukturellen und funktionalen Aufbaus von Synapsen wichtig für das Verständnis von neuronaler Entwicklung sowie von Lernund Gedächtnisprozessen. Synapsen werden durch eine asymmetrische Verbindung von zwei hochspezialisierten Membranen gebildet: An der präsynaptischen aktiven Zone fusionieren mit Transmittern gefüllte Vesikel, während Transmitterrezeptoren in der gegenüberliegenden postsynaptischen Dichte dieses Signal wahrnehmen. Durch genetische Analysen wurde gezeigt, dass Matrixproteine der aktiven Zone verschiedener Familien wichtig für die schnelle Vesikelfusion sind. Es wird angenommen, dass diese Proteine zu synaptischer Stabilität und dem Aufbau von Synapsen beitragen. Das Labor von Stephan Sigrist hat in einer Kollaboration mit dem Labor von Erich Buchner in der Vergangenheit gezeigt, dass das große Gerüstprotein Bruchpilot (Brp) essentiell für sowohl die strukturelle und funktionale Intaktheit von aktiven Zonen als auch für synaptische Plastizität in Drosophila melanogaster ist. Im Zuge dieser Doktorarbeit wurden mehrere Kandidatenproteine untersucht, die vermutlich eine Rolle in prä- und postsynaptischer Funktionen spielen, was folgendermaßen zusammengefasst werden kann: 1. DREP-2 (DFF45 related protein 2) wurde von Dr. Manuela Schmidt durch Koimmunpräzipitationen mit Anti-Brp Antikörpern gefunden (unveröffentlichte Daten). Mutanten und Antikörper für die weitere Untersuchung von DREP-2 wurden im Zuge dieser Doktorarbeit erzeugt. Die Ergebnisse aus Hefe-Zwei-Hybrid Versuchen legen nahe, dass DREP- 2 mit Dynein light chain 2 interagieren könnte, während in vivo Bildgebung darauf hindeutet, dass DREP-2 in bidirektionalen axonalen Transport involviert sein könnte. 2. Koimmunpräzipitations- und Pulldown-Experimente ließen den Schluss zu, dass das ARFGAP-Protein (ADP-ribosylation factor (ARF)-directed GTPase activating proteins (GAPs)) GIT (G-protein coupled receptor kinase interacting protein) mit dem mit Endozytose assoziierten Protein Stoned B (StnB) interagieren könnte. Elektronenmikroskopie der neuromuskulären Synapse von Larven im dritten Larvalstadium, die mutant für das dgit-Gen sind, zeigte eine Akkumulation von großen Vesikeln und Membran-Zwischenprodukten sowie eine verringerte Vesikeldichte. Zwei der Phänotypen, die Akkumulation großer Vesikel und der Membran-Zwischenprodukte, konnten durch die Expression von Drosophila GIT (DGIT) oder menschlichem GIT im dgit-mutanten Hintergrund teilweise ausgeglichen werden. Darüberhinaus wurde über Immunofluoreszenz deutlich, dass die dgit-Mutante eine spezifisch reduzierte Menge an StnB enthält, was durch die Expression von DGIT teilweise ausgeglichen werden konnte. Diese Ergebnisse unterstützen die Vorstellung sehr, dass DGIT mit StnB interagiert.. StnB spielt eine Rolle bei der Regulierung von Vesikelgrößen, Endozytose und der Wiederverwertung von synaptischen Vesikeln. Darüberhinaus zeigen dgit Mutanten Hinweise auf eine fehlerhafte Lokalisierung des präsynaptischen Proteins Brp relativ zu dem postsynaptischen Protein GluRIID, was furch die Expression von DGIT oder menschlichem GIT im dgit-mutanten Hintergrund ausgeglichen werden konnte, nicht jedoch durch StnB. Diese Ergebnisse legen den Schluss nahe, dass GIT einerseits eine Rolle bei der Regulierung der Endozytose synaptischer Vesikel spielt aber möglicherweise auch eine strukturelle Funktion beim Aufbau von Synapsen hat. 3. Djm-1 ist ein genetischer Lokus, der geistige Behinderung bei menschlichen Patienten hervorruft, wenn er mutiert vorliegt. Als ersten Schritt in Richtung eines Verständnisses der mechanistischen Rolle von DJM-1, wurde Genetik in Drosophila durchgeführt, um die Funktion von DJM-1 zu untersuchen. Die in dieser Doktorarbeit erzeugte djm-1 Mutante zeigte jedoch bisher keinen anomalen Phänotyp im Nervensystem
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Engelhardt, [geb Christiansen] Frauke. "Synaptic Connectivity in the Mushroom Body Calyx of Drosophila melanogaster." Doctoral thesis, 2013. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85058.
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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 calyxSynaptische Plastizität an den präsynaptischen Spezialisierungen von Neuronen sind nach allgemeinem Verständnis die Grundlage für Lern- und Gedächtnisprozesse. Kenyon Zellen sind die intrinsischen Zellen des Zentrums für olfaktorisches Lernen im Gehirn von Arthropoden – den Pilzkörper Neuropilen. An den Präsynapsen der Kenyon Zellen wird eine olfaktorische Gedächtnisspur vermutet. Im Kalyx, einer Substruktur der Pilzkörper, erhalten die Kenyon Zell Dendriten ihren olfaktorischen Input durch Projektionsneurone. Ihre Präsynapsen wiederum befinden sich ausschließlich in ihren axonalen Kompartimenten außerhalb des Kalyx, nämlich in den Loben der Pilzkörper. Mit Hilfe von hochauflösenden bildgebenden Techniken und neuen transgenen Methoden, ist es uns in der Fruchtfliege Drosophila melanogaster gelungen, Kenyon Zell Präsynapsen im Kalyx zu identifizieren. Diese Präsynapsen enthalten synaptische Vesikel, die nach Stimulation ihren Inhalt freisetzen können. Sie weisen noch weitere Gemeinsamkeiten mit den meisten anderen Präsynapsen auf: Ihre Aktiven Zonen, die Orte der Transmitterfreisetzung, enthalten die Proteine Bruchpilot und Syd-1. Diese sind Teil der Zytomatrix an der Aktiven Zone, ein Proteingerüst das Endo- und Exozytose der synaptischen Vesikel kontrolliert. Die Präsynapsen im Kalyx wurden in γ- and α/β-Typ Kenyon Zellen aber nicht in α/β-Typ Kenyon Zellen gefunden. Die neu identifizierten Kenyon Zell Präsynapsen beherbergen potentiell eine Gedächtnisspur für olfaktorisch assoziatives Lernen. Möglicherweise wird im olfaktorischen Nervensystem von Fruchtfliegen rücklaufende neuronale Aktivität benötigt, um Gedächtnis abzurufen, so wie es auch für Säuger beschrieben ist. Darüber hinaus zeigen wir synaptische Plastizität im Kalyx. Dies ist die erste Beschreibung überhaupt von synaptischer Plastizität im zentralen Nervensystem von Drosophila melanogaster. Das Volumen des Kalyx kann sich als Antwort auf äußere Einflüsse verändern. Genauso auch Größe und Anzahl der Mikroglomeruli, Substrukturen des Kalyx, in denen Projektionsneurone und Kenyon Zellen aufeinander treffen. Wir untersuchten die Synapsen in Mikroglomeruli detailliert, mithilfe von neuen transgenen Methoden, die es erlauben, präsynaptische Aktive Zonen sowie Postsynaptische Spezialisierungen zu visualisieren. Mittels Beeinträchtigung der Kommunikation zwischen Projektionsneuronen und Kenyon Zellen, konnten wir synaptische Plastizität in Mikroglomeruli zeigen. Projektionsneurone, die nicht in der Lage waren, Aktionspotentiale zu erzeugen, kompensierten ihre funktionelle Einschränkung durch den vermehrten Einbau von Aktiven Zonen in Mikroglomeruli. Außerdem produzierten sie mehr und vergrößerte Mikroglomeruli. Unsere Daten zeigen deutlich eine aktivitätsinduzierte Veränderung des olfaktorischen neuronalen Netzes, sowie strukturelle synaptische Plastizität im Kalyx
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Kisiel, Marta. "Analysis of Myosin VI in Drosophila melanogaster Synaptic Function and Development." Thesis, 2013. http://hdl.handle.net/1807/43617.
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Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is the only member of the myosin superfamily of actin-based motor proteins known to move towards the minus ends of actin filaments. In vitro studies demonstrate that Myosin VI has the ability to perform distinct functions as a cargo transporter and anchor in the cell, however which of these roles Myosin VI plays in the nervous system has yet to be determined. A locomotor defect, observed as sluggish movement in severe jar mutant larvae, was confirmed by behavioural assays. As this can indicate problems at the neuromuscular synapse, microscopy and electrophysiology were used to investigate neuromuscular junction (NMJ) structure and function in jar loss of function mutants of varying severity. Confocal imaging studies revealed a decrease in NMJ length, a reduction in bouton number per NMJ, alterations to the microtubule cytoskeleton and mislocalization of the synaptic vesicle protein Synaptotagmin in jar mutant boutons. FM dye labeling was consistent with the immunostaining data revealing vesicles endocytosed following electrical stimulation occupy the bouton centre in jar mutants. The data is indicative of a function for Myosin VI in maintaining proper peripheral vesicle localization. Electrophysiological experiments revealed a role for Myosin VI in basal synaptic transmission, with a reduction in low frequency nerve-evoked responses and spontaneous release in severe jar mutants. Changes in short-term synaptic plasticity were also observed in Myosin VI mutants by using both paired-pulse experiments to examine release probability and high-frequency stimulation paradigms to recruit vesicles from different functional pools. Taken together, the data suggest that Myosin VI functions as an anchor to peripherally localize vesicles within the bouton enabling their efficient release during nerve stimulation. Synaptic vesicles are mobile at the Drosophila NMJ; thus if Myosin VI is acting as a vesicle tether, it would normally be expected to restrain vesicle mobility at the synapse. FRAP analysis revealed a significant increase in synaptic vesicle mobility in jar mutant boutons. This study elucidates novel roles for Myosin VI function in the nervous system via regulation of the synaptic microtubule architecture and localization of synaptic vesicles within the nerve terminal.
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Bilz, Florian. "Optical Analysis of Synaptic Plasticity Underlying Associative Learning in Drosophila melanogaster." Doctoral thesis, 2018. http://hdl.handle.net/11858/00-1735-0000-002E-E4C2-D.
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Schmidt, Manuela [Verfasser]. "Characterization of synaptic protein complexes in Drosophila melanogaster / submitted by Manuela Schmidt." 2006. http://d-nb.info/981836658/34.
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Silva, Marta Contreiras da. "In vivo mechanisms of synaptic bouton formation: Dissecting the role of the exocyst." Master's thesis, 2018. http://hdl.handle.net/10362/53054.
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RESUMO: A regulação de estruturas sinápticas é crítica para o normal funcionamento neuronal. A morfologia neuronal é geneticamente determinada, mas pode ser modificada por alterações de actividade sináptica, um processo chamado plasticidade estrutural. Botões sinápticos são especializações presinápticas conservadas onde se localizam as sinapses. Apesar da sua importância, os mecanismos que regulam a sua formação são desconhecidos. A junção neuromuscular de Drosophila melanogaster é uma sinapse estereotipada e bem caracterizada, onde plasticidade estrutural pode ser induzida. Após indução, novos botões emergem rapidamente, levando a um aumento da superfície neuronal, mas se este processo requer adição de membrana ou simplesmente rearranjo ainda não é conhecido.
O exocisto é um complexo de várias subunidades que se liga a vesículas secretórias e transporta-as para um local específico da membrana plasmar, interagindo com diversos efectores do citoesqueleto. Tendo isto em conta, questionámo-nos se este complexo teria um papel na formação de botões. Usando um protocolo que induz a formação de novos botões em resposta a actividade, com RNAi contra cada uma das subunidades ou mutações, tentámos saber se alguma das subunidades contribuía para a formação de botões e se a eliminação de cada uma das subunidades teria efeitos diferentes no processo. Este trabalho permitiu concluir que as subunidades estão presentes nos novos botões, e que Sec3 parece ser importante para o processo, enquanto que as larvas de Sec6-IR demonstra um fenótipo locomotor observável. Duas outras subunidades, Sec8 e Exo70, são promissores para estarem envolvidos no processo de formação de novos botões.
Compreender este mecanismo através da formação de botões pode informar-nos acerca da manipulação do número de estruturas sinápticas. Considerando que as doenças neurodegenerativas são usualmente caracterizadas por perda ou simplificação de neurónios, uma alternativa ao aumento do número de neurónios seria estimular vias que resultassem num aumento de estruturas sinápticas.ABSTRACT: Regulation of synaptic structure is critical for proper neuronal function. Neuronal morphology is genetically determined but can be modified by changes in synaptic activity, a process named neuronal structural plasticity. Synaptic boutons are conserved presynaptic specializations where synapses are located. Despite its importance, the mechanisms behind their formation are not fully understood. The neuromuscular junction of the Drosophila melanogaster is a well-characterized stereotyped synapse, where acute structural plasticity can be induced. Upon induction, new boutons emerge quickly as rounded structures, leading to an increase in neuronal surface, but whether this process requires membrane addition or simply a rearrangement remains elusive. The exocyst is a multi-subunit complex that is involved in a variety of mechanisms. It targets secretory vesicles to a specific place at the membrane and interacts with cytoskeletal components. Given this, we asked whether this complex plays a role in activity-dependent bouton formation. By using a protocol that induces new bouton formation in response to activity, with RNAi against each of the subunits or mutations, we assess whether each of the subunits contributes to this process and whether knock down of each of the subunits has different effects. We found that exocyst subunits are present in new synaptic boutons, and that Sec3 appears to be important for this process, while Sec6-IR larvae show a strong locomotor phenotype. Two other subunits, Sec8 and Exo70 may also be involved in the formation of new synaptic boutons, but further analysis will be required. Understanding the mechanism by which activity-dependent bouton formation occurs can provide insights onto how to manipulate the number of synaptic structures. Considering that neurodegenerative diseases are usually characterized by neuronal simplification and loss, an alternative to increasing the number of neurons would be to stimulate pathways that result in increased number of synaptic structures, perhaps ameliorating the function.
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Lee, Yu-Tao, and 李侑道. "Interval between burst of stimuli modulate synaptic plasticity in neuromuscular junction of Drosophila melanogaster larvae." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/17418408012489562334.
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碩士國立清華大學
分子醫學研究所
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Experiments at different levels of neuronal system have shown that the interval between stimuli can affect neuronal system, for example, learning performance or neuronal excitability. However, the effect of different interval between stimuli is not clear in synaptic level. In the present study, we have used the neuromuscular junction (NMJ) of Drosophila third instar larvae and changed the interval between bursts to explore the role of inter-burst interval (IBI) in synaptic plasticity. When calcium concentration in buffer is 0.2mM, the amplitude of evoked junctional potential (EJP ) is suppressed after giving tetanus stimuli with IBI = 0 or 3.25 sec. When IBI is 1.25 sec, between 0 sec and 3.25 sec, the amplitude of EJP is not depressed after tetanus. The effects of different IBI on the synaptic plasticity are the same when the length of tetanus or the number of bursts is changed. Changing the calcium concentration in buffer from 0.2mM to 0.4mM affects the interval tuning at synaptic plasticity. At [Ca2+] = 0.4 mM, tetanus with IBI = 3.25 sec has no depression effect on EJP amplitude, but IBI = 1.25 sec suppresses EJP amplitude after tetanus. We have used rut2080, a mutant with reduced cAMP, to investigate the role of cAMP cascade in IBI tuning synaptic plasticity. At [Ca2+] = 0.2 mM, the suppressed effect on EJP amplitude when giving tetanus with IBI = 3.25 sec disappears. Besides the long term effect of synaptic plasticity, different IBI also affects the synaptic plasticity during tetanus. Thus, these results show that the length of IBI might be an important factor affecting synaptic plasticity at NMJ, and NMJ might discriminate different IBI through Ca2+ and cAMP cascade.
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Marek, Kurt W. "Molecular and phenomenological characterization of synaptic homeostasis at the Drosophila neuromuscular junction /." 2004. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3136062.
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Edwards, Tara N. "GLIAL DEVELOPMENT, SYNAPTIC PLASTICITY AND NEUROTRANSMITTER RECYCLING IN THE VISUAL SYSTEM OF THE FRUIT FLY DROSOPHILA MELANOGASTER." 2010. http://hdl.handle.net/10222/13142.
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The visual system of the fruit fly is ideal for studying the association between neurons and glia. These interact during the morphogenesis of brain neuropiles and, in the adult, work together to maintain an ideal environment for neuronal function. In this thesis I characterise the pupal metamorphosis and adult structure of glia in the optic lobe’s lamina and medulla neuropiles. Photoreceptor axons from the fly’s compound eyes terminate at locations within these neuropiles that allow them intimate contact with glia. Some neuropile glia take up and inactivate the neurotransmitter histamine after its release at photoreceptor synapses. A shuttle pathway between the glia and photoreceptors then transports inactivated histamine back to photoreceptors for reuse. The gene CG12120 encodes the protein Tan, which liberates recycled histamine within the photoreceptor cytoplasm, it is then pumped into vesicles for re-release. Histamine, however, is not exclusive to the visual system. A system of glial barriers in the lamina, and around the brain, controls the movement of histamine between neuropiles as well as between the body and the brain. How histamine is reciprocally transported between photoreceptors and glia remains unknown despite attempts to uncover candidate transporters. Photoreceptor-specific capitate projections form as invaginations from neuropile glia into photoreceptor terminals. The transmitter needs of the photoreceptor appear to dictate the dynamic structure of capitate projections, which change in shape and number after perturbations that affect terminal location, synapse number or histamine release at the photoreceptor. This dynamism suggests that capitate projections play an important role, not only in recycling synaptic vesicles, but also in recycling histamine.
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Knapek, Stephan. "Synapsin and Bruchpilot, two synaptic proteins underlying specific phases of olfactory aversive memory in Drosophila melanogaster." Doctoral thesis, 2010. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-49726.
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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 ARMGedächtnis ist ein dynamischer Prozess. In der Zeit kurz nach seiner Bildung ist es instabil und anfällig gegen amnestische Störungen, dann wird es schrittweise konsolidiert und schließlich resistent gegenüber retrogradem Gedächtnisverlust (McGaugh, 2000). Auch das assoziative olfaktorische Gedächtnis der Fruchtfliege Drosophila melanogaster zeigt diese Merkmale. Nach einem einzelnen assoziativen Training, in welchem ein Duft mit elektrischen Stromstößen gepaart wird, bilden die Fliegen ein aversives Duftgedächtnis, welches über mehrere Stunden anhält und aus qualitativ unterschiedlichen Komponenten besteht (Quinn et al., 1974; Tully and Quinn, 1985). Diese Komponenten können zum Beispiel durch Mutationen, die zugrunde liegenden neuronalen Verknüpfungen oder durch ihre Anfälligkeit für amnestische Behandlungen unterschieden werden (Dubnau and Tully, 1998; Isabel et al., 2004; Keene and Waddell, 2007; Masek and Heisenberg, 2008; Xia and Tully, 2007). Eine gegen amnestische Behandlungen, wie beispielsweise Kälte-induzierte Betäubung, anfällige Komponente beherrscht das frühe Gedächtnis, zerfällt jedoch schnell (Margulies et al., 2005; Quinn and Dudai, 1976). Diese wird deshalb Anästhesie-sensitives Gedächtnis genannt (anesthesia-sensitive memory [ASM]). Im Gegensatz dazu baut sich eine konsolidierte Komponente erst langsam in den folgenden Stunden nach dem Training auf, hält stattdessen jedoch länger an (Margulies et al., 2005; Quinn and Dudai, 1976). Diese Komponente ist resistent gegenüber Kälte-induzierter Anästhesie und wird deshalb als ARM (anesthesia-resistant memory) bezeichnet. In der vorliegenden Arbeit konnte ich zeigen, dass das konsolidierte ARM bereits mit deutlich weniger starken Elektroschocks im Training gebildet wird als das instabile ASM. ARM und ASM unterliegen unterschiedliche molekulare und/oder neuronale Prozesse. Während in einer Mutante für das radish Gen selektiv ARM beeinträchtigt ist, werden andere Gene wie zum Beispiel amnesiac oder rutabaga ausschließlich für ASM benötigt (Dudai et al., 1988; Folkers et al., 1993; Isabel et al., 2004; Quinn and Dudai, 1976; Schwaerzel et al., 2007; Tully et al., 1994). Die beiden letzteren sind Teil des cAMP Signalweges, welcher vermutlich hauptsächlich die cAMP abhängige Protein-Kinase A (PKA) aktiviert (Levin et al., 1992). Hier zeige ich, dass eine Null-Mutante für das evolutionär konservierte Phosphoprotein Synapsin einen selektiven Defekt in ASM hat. Weitere Experimente lassen vermuten, dass Synapsin als Effektor stromabwärts der cAMP/PKA Kaskade wirkt. Ähnlich wie bei Drosophila spielt Synaspin auch in Vertebraten eine Rolle in unterschiedlichen Lernparadigmen (Gitler et al., 2004; Silva et al., 1996). Auch in der Meeresschnecke Aplysia wurde eine PKA abhängige Phosphorylierung von Synapsin als Mechanismus für die Regulierung von Neurotransmitterausschüttung und Kurzzeitplastizität vorgeschlagen (Angers et al., 2002; Fiumara et al., 2004). Synapsin wird für die Bildung eines Reserve-Pools von Vesikeln an der Präsynapse und für die Aufrechterhaltung der Vesikelausschüttung speziell bei anhaltender, hochfrequenter Stimulation von Nervenzellen benötigt (Akbergenova and Bykhovskaia, 2007; Li et al., 1995; Pieribone et al., 1995; Sun et al., 2006). Im Gegensatz dazu wird Bruchpilot, ein Protein der aktiven Zone und homolog zu den ELKS/CAST Proteinen bei Säugern (Wagh et al., 2006), haupsächlich für sofortige Vesikelausschüttung gebraucht (Kittel et al., 2006). Bei wiederholter Stimulation an Motorneuronen einer bruchpilot Mutante ist die akute Vesikelausschüttung stark vermindert, während die darauf folgende andauernde Ausschüttung noch immer möglich ist (Kittel et al., 2006). Dazu passend beeinträchtigt eine künstliche Verminderung des Bruchpilot-Proteins die Ansammlung von Ca2+ Kanälen an den aktiven Zonen, sowie die Bildung von elektronendichten Strukturen (T-bars) an den präsynaptischen Endigungen. Deshalb akkumulieren weniger Vesikel des “readily-releasable” Pools an den Ausschüttungsstellen und die Ausschüttungswahrscheinlichkeit ist stark vermindert (Kittel et al., 2006; Wagh et al., 2006). In dieser Arbeit zeige ich zum ersten Mal, dass Bruchpilot für aversives olfaktorisches Gedächtnis benötigt wird. Der Ort an dem Bruchpilot hierfür gebraucht wird sind die Kenyon-Zellen des Pilzkörpers, die olfaktorischen Interneuronen zweiter Ordnung in Drosophila. Desweiteren zeige ich, dass die Funktion von Bruchpilot selektiv für das konsolidierte ARM ist. Da Synapsin spezifisch für das labile ASM benötigt wird, können diese beiden olfaktorischen Gedächtniskomponenten durch verschiedene synaptische Proteine getrennt werden, und zwei unterschiedliche Arten der Neurotransmitterausschüttung (abhängig von hoch- oder niedrig-frequenter Stimulation) könnten ASM und ARM auseinander halten
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Mende, Michael [Verfasser]. "Analysing the role of short stop during the formation of synaptic terminals in Drosophila melanogaster / Michael Mende." 2004. http://d-nb.info/971998396/34.
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Halder, Partho. "Identification and characterization of synaptic proteins of Drosophila melanogaster using monoclonal antibodies of the Wuerzburg Hybridoma Library." Doctoral thesis, 2011. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-67325.
Full textAbstract:
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 structuresFür einen Großteil der Proteine, die im menschlichen Gehirn exprimiert werden, ist lediglich die Primärstruktur aus dem Genomprojekt bekannt. Proteine, die in der Evolution konserviert wurden, können in genetischen Modellsystemen wie Drosophila untersucht werden. In dieser Doktorarbeit werden monoklonale Antikörper (mAk) aus der Würzburger Hybridoma Bibliothek produziert und charakterisiert, mit dem Ziel, die erkannten Proteine zu identifizieren. Der mAk ab52 wurde als IgM typisiert, das auf Western Blots ein zytosolisches Protein von Mr ~110 kDa erkennt. Das Antigen wurde durch zwei-dimensionale Gelelektrophorese (2DE) als einzelner Fleck aufgelöst. Massenspektrometrische Analyse dieses Flecks identifizierte dass EPS-15 (epidermal growth factor receptor pathway substrate clone 15) als viel versprechenden Kandidaten. Da für einen anderen mAk aus der Bibliothek, aa2, bereits bekannt war, dass er EPS-15 erkennt, wurden die Western-Blot-Signale der beiden Antikörper nach 1D und 2D Trennungen von Kopfhomogenat verglichen. Die Ähnlichkeit der beiden Muster deuteten darauf hin, dass beide Antigene dasselbe Protein erkennen. Das Fehlen des Wildtyp-Signals in homozygoten Eps15 Mutanten in einem Western Blot mit mAk ab52 bestätigten schließlich, dass EPS-15 das Antigen zu mAk ab52 darstellt. Demnach erkennen beide mAk, aa2 und ab52, das Drosophila Homolog zu EPS-15. Da mAk aa2 ein IgG ist, dürfte er für Anwendungen wie Immunpräzipitation (IP) besser geeignet sein. Er wurde daher bereits bei der Developmental Studies Hybridoma Bank (DSHB) eingereicht, um ihn der ganzen Forschergemeinde leicht zugänglich zu machen. Der mAk na21 wurde ebenfalls als IgM typisiert. Er erkennt ein Membran assoziiertes Antigen von Mr ~10 kDa auf Western Blots. Aufgrund der Membranassoziierung des Proteins war es nicht möglich, es in 2DE aufzulösen und da es sich um ein IgM handelt, war eine Anreicherung des Antigens mittels IP nicht erfolgreich. Vorversuche zur biochemischen Reinigung des endogenen Proteins aus Gewebe waren Erfolg versprechend, konnten aber aus Zeitmangel nicht abgeschlossen werden. Daher erscheint eine biochemische Reinigung des Proteins für eine Identifikation durch Massenspektrometrie möglich. Eine Reihe weiterer mAk wurden hinsichtlich ihrer Färbemuster auf Gefrierschnitten und in Ganzpräparaten von Drosophila Gehirnen untersucht. Allerdings färbten viele dieser mAk sehr wenige Strukturen im Gehirn, so dass nur eine sehr begrenzte Menge an Protein als Startmaterial verfügbar wäre. Da diese Antikörper keine Signale auf Western Blots produzierten und daher eine Anreicherung des Antigens durch elektrophoretische Methoden ausschlossen, wurde keine Reinigung versucht. Andererseits macht die spezifische Lokalisation dieser Proteine sie hoch interessant für eine weitere Charakterisierung, da sie eine besonders spezialisierte Rolle in der Entwicklung oder für die Funktion von neuralen Schaltkreisen, in denen sie vorkommen, spielen könnten. Die Reinigung und Identifikation solcher Proteine mit niedrigem Expressionsniveau würde neue Methoden der Anreicherung der gefärbten Strukturen erfordern