Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

drosophila-larval-nmj-immunohistochemistry

<em>Drosophila</em> Larval NMJ Immunohistochemistry

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Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.

wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila.

Wunschdenken kodiert f&#xFC;r ein BMP-Typ-II-Rezeptor, die synaptische Wachstum in Drosophila regelt.

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability.

Mitglieder der Synaptobrevin / Vesikel-assoziiertes Membranprotein (VAMP) Familie in Drosophila funktionell austauschbar in vivo Freisetzung von Neurotransmittern und die Lebensf&#xE4;higkeit der Zellen.

Das BMP-Homolog Gbb bietet eine retrograde Signal, das synaptische Wachstum regelt an der neuromuskul&#xE4;ren Synapse Drosophila.

We show that the BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those we observe in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, we find that blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth.

The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction.

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction.

Retrograde Kontrolle der synaptischen &#xDC;bertragung durch postsynaptische CaMKII an der neuromuskul&#xE4;ren Synapse Drosophila.

Highwire (Hiw), a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Here we show that Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses.

Highwire regulates presynaptic BMP signaling essential for synaptic growth.

Highwire regelt pr&#xE4;synaptischen BMP-Signalweg essentiell f&#xFC;r synaptische Wachstum.

Autosomal-dominant pure hereditary spastic paraplegia (AD-HSP) is characterized by the degeneration of long axons in corticospinal tracts and dorsal columns, resulting in spasticity and difficulty walking. Mutations in the SPG4 gene product spastin are the predominant genetic lesions associated with this inherited disease. In this issue, Orso et al. examine and reconcile existing Drosophila mutants of spastin and generate a new model for HSP by overexpression of a fly spastin transgene that carries a mutation prevalent in human AD-HSP (see the related article beginning on page 3026). Expression of this mutant spastin protein produces pathology in flies reminiscent of the human disease, including adult locomotion defects, in addition to causing aberrant synaptic morphology and altered microtubule stability. Both movement and synaptic defects in fly mutants were ameliorated by treatment with the microtubule-modifying agent vinblastine. The results are consistent with disease-causing mutations in human spastin producing dominant-negative proteins and confirm the usefulness of Drosophila genetic techniques to understand HSP and other neurodegenerative diseases.

All neuropathies great and small.

Autosomal-dominante reine Heredit&auml;re Spastische Paraplegie (AD-HSP) zeichnet sich durch die Degeneration der langen Axone im corticospinal Tracts und dorsalen Spalten, was Spastizit&auml;t und Schwierigkeiten beim gehen. Mutationen in den SPG4 gen Produkt Spastin sind die vorherrschenden genetischen L&auml;sionen dieser vererbten Krankheit zugeordnet. In dieser Ausgabe Orso Et Al. untersuchen und vorhandene Drosophila-Mutanten von Spastin zu vers&ouml;hnen und generieren ein neues Modell f&uuml;r HSP durch eine &Uuml;berexpression von eine Transgene fliegen Spastin, die eine Mutation im menschlichen AD-HSP tr&auml;gt (siehe Verwandte Artikel Anfang Seite 3026). Ausdruck dieses Proteins mutant Spastin produziert Pathologie in fliegen erinnert an die menschliche Krankheit, einschlie&szlig;lich Erwachsener Fortbewegung M&auml;ngel, neben verursacht aberrante synaptische Morphologie und Microtubule Stabilit&auml;t ver&auml;ndert. Bewegung und synaptische Defekte in fliegen Mutanten wurden durch die Behandlung mit dem &Auml;ndern von Microtubule Agent Vinblastin einged&auml;mmt. Die Ergebnisse stehen im Einklang mit krankheitsverursachende Mutationen im menschlichen Spastin Produktion Dominant-negativen Proteine und best&auml;tigen Sie die N&uuml;tzlichkeit der Taufliege genetische Techniken, HSP und anderen neurodegenerativen Erkrankungen zu verstehen.

Alle Neuropathien, gro&szlig; und klein.

A molecular understanding of synaptogenesis is a critical step toward the goal of understanding how brains "wire themselves up," and then "rewire" during development and experience. Recent genomic and molecular advances have made it possible to study synaptogenesis on a genomic scale. Here, we describe the results of a screen for genes involved in formation and development of the glutamatergic Drosophila neuromuscular junction (NMJ). We screened 2185 P-element transposon mutants representing insertions in approximately 16% of the entire Drosophila genome. We first identified recessive lethal mutants, based on the hypothesis that mutations causing severe disruptions in synaptogenesis are likely to be lethal. Two hundred twenty (10%) of all insertions were homozygous lethal. Two hundred five (93%) of these lethal mutants developed at least through late embryogenesis and formed neuromusculature. We examined embryonic/larval NMJs in 202 of these homozygous mutants using immunocytochemistry and confocal microscopy. We identified and classified 88 mutants with altered NMJ morphology. Insertion loci in these mutants encode several different types of proteins, including ATP- and GTPases, cytoskeletal regulators, cell adhesion molecules, kinases, phosphatases, RNA regulators, regulators of protein formation, transcription factors, and transporters. Thirteen percent of insertions are in genes that encode proteins of novel or unknown function. Complementation tests and RT-PCR assays suggest that approximately 51% of the insertion lines carry background mutations. Our results reveal that synaptogenesis requires the coordinated action of many different types of proteins--perhaps as much as 44% of the entire genome--and that transposon mutageneses carry important caveats that must be respected when interpreting results generated using this method.

Genome-wide P-element screen for Drosophila synaptogenesis mutants.

Ein molekularen Verst&auml;ndnis der Synaptogenese ist ein entscheidender Schritt auf das Ziel zu verstehen, wie Gehirne &quot;Draht selbst oben&quot;, und dann &quot;rewire&quot;, w&auml;hrend Entwicklung und Erfahrung. Die j&uuml;ngsten Fortschritte der genomischen und Molekulare haben Synaptogenese auf genomischer Ebene zu studieren erm&ouml;glicht. Hier beschreiben wir die Ergebnisse eines Bildschirms f&uuml;r Gene, die Entstehung und Entwicklung von Glutamat Drosophila neuromuskul&auml;rer Synapsen (NMJ) beteiligt. Wir geschirmt 2185 P-Elements Transposon Mutanten, die Einf&uuml;gungen in etwa 16 % des gesamten Genoms Drosophila darstellt. Wir haben zun&auml;chst identifiziert, rezessive letale Mutanten, basierend auf der Hypothese, dass Mutationen verursachen gravierende St&ouml;rungen im Synaptogenese t&ouml;dlich sein d&uuml;rften. Zwei hundert zwanzig (10 %) von allen Einf&uuml;gungen waren homozygot letal. Zwei hundert f&uuml;nf (93 %) diese letale Mutanten entwickelt mindestens durch sp&auml;ten Embryogenese und Neuromusculature gebildet. Wir untersuchten embryonale/larvalen NMJs 202 diese homozygoten Mutanten mit Immunzytochemie und confocal Mikroskopie. Wir identifiziert und klassifiziert 88 Mutanten mit ver&auml;nderten NMJ-Morphologie. Einf&uuml;gung Loci in diese Mutanten codieren, verschiedene Arten von Proteine, einschlie&szlig;lich ATP- und GTPasen, Zytoskelett Regulatoren, Zelladh&auml;sionsmolek&uuml;le, Kinasen, Phosphatasen, RNA-Regulatoren, die Regler der Protein-Formation, Transkriptionsfaktoren und Transporter. Dreizehn Prozent der Einf&uuml;gungen sind Gene, die Proteine des Romans oder unbekannte Funktion codieren. Komplementation Pr&uuml;fungen und RT-PCR-Assays zufolge etwa 51 % der Einf&uuml;gung Linien Hintergrund Mutationen tragen. Unsere Ergebnisse zeigen, dass Synaptogenese erfordert koordinierte Vorgehen der viele verschiedene Arten von Proteinen--vielleicht weniger als 44 % des gesamten Genoms-- und dass die Transposon Mutageneses wichtige Vorsichtsma&szlig;nahmen durchf&uuml;hren, die eingehalten werden m&uuml;ssen, bei der Interpretation der Ergebnisse, die mit dieser Methode generiert.

Genomweiten P-Elements Bildschirm f&uuml;r Drosophila Synaptogenese Mutanten.

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.

Die t&ouml;dliche Motor neurons Krankheit Amyotrophe Lateralsklerose (ALS) teilt einige klinische und pathologische &Uuml;berschneidungen mit Frontotemporale Demenz (FTD), eine fr&uuml;h beginnende Neurodegenerative Erkrankung. Die RNA/DNA-bindenen Proteine verschmolzen in Sarkom (FUS; auch bekannt als TLS) und TAR DNA binding Protein-43 (TDP-43) haben vor kurzem gezeigt worden, um genetisch und krankhaft von famili&auml;ren Formen ALS und FTD zugeordnet werden. Es ist derzeit nicht bekannt, ob Ergebnisse einer St&ouml;rung dieser Proteine in Krankheit durch Mechanismen, die sind unabh&auml;ngig, der normale Protein-Funktion oder &uuml;ber die pathophysiologische St&ouml;rung der molekularen Prozesse, in denen sie beide kritisch sind. Hier berichten wir, dass Drosophila-Mutanten, in dem die Homolog des FUS gest&ouml;rte Ausstellung verringerte sich um Erwachsene Lebensf&auml;higkeit, ist, vermindert lokomotorische Geschwindigkeit und reduziert die Lebensdauer im Vergleich zu Kontrollen. Diese Ph&auml;notypen wurden vollst&auml;ndig von Wildtyp menschliche FUS, aber nicht ALS-assoziierter mutant FUS Proteine gerettet. Eine Mutante der Drosophila-Homolog von TDP-43 hatte &auml;hnliche, aber noch versch&auml;rft, Defizite. Durch Kreuz-Rettung-Analyse zeigten wir, dass FUS zusammen mit und stromabw&auml;rts von TDP-43 in einen gemeinsamen genetischen Weg in Neuronen gehandelt. Dar&uuml;ber hinaus fanden wir, dass diese Proteine, die einander in einem RNS-abh&auml;ngige-Komplex zugeordnet. Unsere Ergebnisse belegen, dass FUS und TDP-43 in Vivo zusammen funktionieren und schlagen vor, da&szlig; Molekulare Wege erfordern die kombinierten Aktivit&auml;ten beider dieser Proteine ALS und FTD gest&ouml;rt werden k&ouml;nnen.

ArticleTotal : 3	Year
Publication title Cited by 114	2009
Publication title Cited by 25	2009
Publication title Cited by 29	2009

Article	Year
wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron\| PubMed ID: 11856529	2002
Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 12364587	2002
The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron\| PubMed ID: 12873382	2003
Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction. Neuron\| PubMed ID: 12873383	2003
Highwire regulates presynaptic BMP signaling essential for synaptic growth. Neuron\| PubMed ID: 15046722	2004
All neuropathies great and small. The Journal of clinical investigation\| PubMed ID: 16276409	2005
Genome-wide P-element screen for Drosophila synaptogenesis mutants. Journal of neurobiology\| PubMed ID: 16408305	2006
The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span. The Journal of clinical investigation\| PubMed ID: 21881207	2011

Brian D. McCabe

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