Two Algorithms for High-throughput and Multi-parametric Quantification of &lt;em&gt;Drosophila&lt;/em&gt; Neuromuscular Junction Morphology

Nijhof, Bonnie; Eidhof, Ilse; Wolf, Louis; Gooyert, Jolanda M. Scheffer-de; Monedero, Ignacio; Torroja, Laura; Laak, Jeroen van der; Schenck, Annette

doi:10.3791/55395

Cited by 7 publications

(7 citation statements)

References 28 publications

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“…Automated-thresholding was performed to binarize punctate images (i.e. for synapse markers, EAAT2 or p-Ezrin) using a "Moments" algorithm, as is well suited to punctate analysis 4,100,101 . GFAP images were binarized using "Li" based automated thresholding.…”

Section: Mapping Image Analysismentioning

confidence: 99%

Nanostructural Diversity of Synapses in the Mammalian Spinal Cord

Broadhead

Bonthron

Arcinas

et al. 2020

Sci Rep

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functionally distinct synapses exhibit diverse and complex organisation at molecular and nanoscale levels. Synaptic diversity may be dependent on developmental stage, anatomical locus and the neural circuit within which synapses reside. Furthermore, astrocytes, which align with pre and postsynaptic structures to form 'tripartite synapses', can modulate neural circuits and impact on synaptic organisation. In this study, we aimed to determine which factors impact the diversity of excitatory synapses throughout the lumbar spinal cord. We used PSD95-eGFP mice, to visualise excitatory postsynaptic densities (pSDs) using high-resolution and super-resolution microscopy. We reveal a detailed and quantitative map of the features of excitatory synapses in the lumbar spinal cord, detailing synaptic diversity that is dependent on developmental stage, anatomical region and whether associated with VGLUT1 or VGLUT2 terminals. We report that PSDs are nanostructurally distinct between spinal laminae and across age groups. PSDs receiving VGLUT1 inputs also show enhanced nanostructural complexity compared with those receiving VGLUT2 inputs, suggesting pathway-specific diversity. Finally, we show that PSDs exhibit greater nanostructural complexity when part of tripartite synapses, and we provide evidence that astrocytic activation enhances PSD95 expression. Taken together, these results provide novel insights into the regulation and diversification of synapses across functionally distinct spinal regions and advance our general understanding of the 'rules' governing synaptic nanostructural organisation. Neuronal synapses show considerable structural, molecular and functional diversity throughout the nervous system. Their morphology, molecular composition and nanostructural organisation determines synaptic strength and function 1,2 , and must therefore be finely tuned to the circuits within which they operate 3. Synapse morphology may be determined by a number of factors, such as the type of neuron, the anatomical location, the activity state of the network, and development and ageing 4-9. In order to understand the basic logic of different neural circuits, it is pertinent to study the principles of synaptic morphology that underlie the function of neural circuits. Such information may help produce a set of basic 'rules' governing synaptic structure and therefore function. Key scaffolding proteins such as PSD95, one of the most abundantly expressed proteins from the Dlg family, form the core molecular architecture of the synapse 10-13. At a molecular level, PSD95 interacts with a host of different signalling enzymes and neurotransmitter receptors within the postsynaptic density (PSD) to form PSD95-dependent super-complexes (~1.5MDa in weight) 14-17. These super-complexes are heterogeneously distributed within the PSD, forming protein-enriched domains known as nanoclusters (NCs, ~140 nm diameter) 4,5,18,19 , which align with presynaptic release sites to form trans-synaptic nanocolumns 1. Visualising PSD95 enables us to determine the na...

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Section: Mapping Image Analysismentioning

confidence: 99%

Nanostructural Diversity of Synapses in the Mammalian Spinal Cord

Broadhead

Bonthron

Arcinas

et al. 2020

Sci Rep

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“…4A). Morphometric features of individual muscle four NMJs were quantified semiautomatically (53,54). We detected mean values of area, perimeter, and length in Oga D133N and Oga KO NMJs similar to those in controls (Fig.…”

Section: Drosophila Oga Is Required For Habituation Learningmentioning

confidence: 92%

“…Projections of type 1b NMJs at muscle 4 from abdominal segments A2-A4 were visualized with Zeiss Axio Imager Z2 microscope with Apotome. Individual synapses were imaged and quantified using an inhouse developed macro (53,54) in Fiji (version 1.49) (73). NMJ Drosophila Oga is required for habituation learning area, length, number of branches, and number of branching points were analyzed based on discs large labeling, and the number of synaptic boutons was analyzed based on synaptotagmin labeling.…”

Section: Analysis Of Synaptic Morphologymentioning

confidence: 99%

O-GlcNAcase contributes to cognitive function in Drosophila

Muha

Fencková

Ferenbach

et al. 2020

Journal of Biological Chemistry

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O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO, respectively) in Drosophila melanogaster. Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.

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“…Images were obtained with the Zeiss Axio Imager 2 using the ApoTome. Obtained images were analysed with the macros Drosophila _NMJ_Bouton_Morphometrics and Drosophila _NMJ_ Morphometrics macro in FIJI [120, 122, 123]. GraphPad Prism software was used for statistical analysis.…”

Section: Methodsmentioning

confidence: 99%

Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila

Castells-Nobau¹,

Eidhof²,

Fencková³

et al. 2019

PLoS ONE

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FOXP proteins form a subfamily of evolutionarily conserved transcription factors involved in the development and functioning of several tissues, including the central nervous system. In humans, mutations in FOXP1 and FOXP2 have been implicated in cognitive deficits including intellectual disability and speech disorders. Drosophila exhibits a single ortholog, called FoxP, but due to a lack of characterized mutants, our understanding of the gene remains poor. Here we show that the dimerization property required for mammalian FOXP function is conserved in Drosophila . In flies, FoxP is enriched in the adult brain, showing strong expression in ~1000 neurons of cholinergic, glutamatergic and GABAergic nature. We generate Drosophila loss-of-function mutants and UAS-FoxP transgenic lines for ectopic expression, and use them to characterize FoxP function in the nervous system. At the cellular level, we demonstrate that Drosophila FoxP is required in larvae for synaptic morphogenesis at axonal terminals of the neuromuscular junction and for dendrite development of dorsal multidendritic sensory neurons. In the developing brain, we find that FoxP plays important roles in α-lobe mushroom body formation. Finally, at a behavioral level, we show that Drosophila FoxP is important for locomotion, habituation learning and social space behavior of adult flies. Our work shows that Drosophila FoxP is important for regulating several neurodevelopmental processes and behaviors that are related to human disease or vertebrate disease model phenotypes. This suggests a high degree of functional conservation with vertebrate FOXP orthologues and established flies as a model system for understanding FOXP related pathologies.

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Two Algorithms for High-throughput and Multi-parametric Quantification of <em>Drosophila</em> Neuromuscular Junction Morphology

Cited by 7 publications

References 28 publications

Nanostructural Diversity of Synapses in the Mammalian Spinal Cord

Nanostructural Diversity of Synapses in the Mammalian Spinal Cord

O-GlcNAcase contributes to cognitive function in Drosophila

Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila

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