Academic literature on the topic 'Drosophila melanogaster Synaptic Transmission'

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 neuronale<br>The 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

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.

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.

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.

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-jacent<br>Structural 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

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.

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.