Detecting vibrations in digital holographic multiwavelength measurements using deep learning.

Digital holographic multiwavelength sensor systems integrated in the production line on multi-axis systems such as robots or machine tools are exposed to unknown, complex vibrations that affect the measurement quality. To detect vibrations during the early steps of hologram reconstruction, we propose a deep learning approach using a deep neural network trained to predict the standard deviation of the hologram phase. The neural network achieves 96.0% accuracy when confronted with training-like data while it achieves 97.3% accuracy when tested with data simulating a typical production environment. It performs similar to or even better than comparable classical machine learning algorithms. A single prediction of the neural network takes 35 µs on the GPU.

Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor.

Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here, we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior.

Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons.

A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. Here, we examined the role of the astrocytic GABA transporter (GAT) in sleep regulation using Drosophila melanogaster. We found that a hypomorphic gat mutation (gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABAA receptor resistance to dieldrin (RDL) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. We concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila.

Focal adhesion molecules regulate astrocyte morphology and glutamate transporters to suppress seizure-like behavior.

Astrocytes are important regulators of neural circuit function and behavior in the healthy and diseased nervous system. We screened for molecules in Drosophila astrocytes that modulate neuronal hyperexcitability and identified multiple components of focal adhesion complexes (FAs). Depletion of astrocytic Tensin, ß-integrin, Talin, focal adhesion kinase (FAK), or matrix metalloproteinase 1 (Mmp1), resulted in enhanced behavioral recovery from genetic or pharmacologically induced seizure. Overexpression of Mmp1, predicted to activate FA signaling, led to a reciprocal enhancement of seizure severity. Blockade of FA-signaling molecules in astrocytes at basal levels of CNS excitability resulted in reduced astrocytic coverage of the synaptic neuropil and expression of the excitatory amino acid transporter EAAT1. However, induction of hyperexcitability after depletion of FA-signaling components resulted in enhanced astrocyte coverage and an approximately twofold increase in EAAT1 levels. Our work identifies FA-signaling molecules as important regulators of astrocyte outgrowth and EAAT1 expression under normal physiological conditions. Paradoxically, in the context of hyperexcitability, this pathway negatively regulates astrocytic process outgrowth and EAAT1 expression, and their blockade leading to enhanced recovery from seizure.

Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour.

Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca2+). Astrocytic Ca2+ signalling has been proposed to modulate neural circuit activity, but the pathways that regulate these events are poorly defined and in vivo evidence linking changes in astrocyte Ca2+ levels to alterations in neurotransmission or behaviour is limited. Here we show that Drosophila astrocytes exhibit activity-regulated Ca2+ signalling in vivo. Tyramine and octopamine released from neurons expressing tyrosine decarboxylase 2 (Tdc2) signal directly to astrocytes to stimulate Ca2+ increases through the octopamine/tyramine receptor (Oct-TyrR) and the transient receptor potential (TRP) channel Water witch (Wtrw), and astrocytes in turn modulate downstream dopaminergic neurons. Application of tyramine or octopamine to live preparations silenced dopaminergic neurons and this inhibition required astrocytic Oct-TyrR and Wtrw. Increasing astrocyte Ca2+ signalling was sufficient to silence dopaminergic neuron activity, which was mediated by astrocyte endocytic function and adenosine receptors. Selective disruption of Oct-TyrR or Wtrw expression in astrocytes blocked astrocytic Ca2+ signalling and profoundly altered olfactory-driven chemotaxis and touch-induced startle responses. Our work identifies Oct-TyrR and Wtrw as key components of the astrocytic Ca2+ signalling machinery, provides direct evidence that octopamine- and tyramine-based neuromodulation can be mediated by astrocytes, and demonstrates that astrocytes are essential for multiple sensory-driven behaviours in Drosophila.

Activity-dependent regulation of astrocyte GAT levels during synaptogenesis.

Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. We found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABA(B)R1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABA(B)R1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.

Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.

Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.

Analysis of glial cell development and function in Drosophila.

Glial cells are the most abundant cell type in our brains, yet we understand very little about their development and function. An accumulating body of work over the last decade has revealed that glia are critical regulators of nervous system development, function, and health. Based on morphological and molecular criteria, glia in Drosophila melanogaster are very similar to their mammalian counterparts, suggesting that a detailed investigation of fly glia has the potential to add greatly to our understanding of fundamental aspects of glial cell biology. In this article, we provide an overview of the subtypes of glial cells found in Drosophila and discuss our current understanding of their functions, the development of a subset of well-defined glial lineages, and the molecular-genetic tools available for manipulating glial subtypes in vivo.

The CD59 family member Leaky/Coiled is required for the establishment of the blood-brain barrier in Drosophila.

The blood-brain barrier of Drosophila is established by the subperineurial glial cells that encase the CNS and PNS. The subperineurial glial cells are thin, highly interdigitated cells with epithelial character. The establishment of extensive septate junctions between these cells is crucial for the prevention of uncontrolled paracellular leakage of ions and solutes from the hemolymph into the nervous system. In the absence of septate junctions, macromolecules such as fluorescently labeled dextran can easily cross the blood-brain barrier. To identify additional components of the blood-brain barrier, we followed a genetic approach and injected Texas-Red-conjugated dextran into the hemolymph of embryos homozygous for chromosomal deficiencies. In this way, we identified the 153-aa-large protein Coiled, a new member of the Ly6 (leukocyte antigen 6) family, as being crucially required for septate junction formation and blood-brain barrier integrity. In coiled mutants, the normal distribution of septate junction markers such as NeurexinIV, Coracle, or Discs large is disturbed. EM analyses demonstrated that Coiled is required for the formation of septate junctions. We further show that Coiled is expressed by the subsperineurial glial cells in which it is anchored to the cell membrane via a glycosylphosphatidylinositol anchor and mediates adhesive properties. Clonal rescue studies indicate that the presence of Coiled is required symmetrically on both cells engaged in septate junction formation.

Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper.

Ensheathment of axons by glial membranes is a key feature of complex nervous systems ensuring the separation of single axons or axonal fascicles. Nevertheless, the molecules that mediate the recognition and specific adhesion of glial and axonal membranes are largely unknown. We use the Drosophila midline of the embryonic central nervous system as a model to investigate these neuron glia interactions. During development, the midline glial cells acquire close contact to commissural axons and eventually extend processes into the commissures to wrap individual axon fascicles. Here, we show that this wrapping of axons depends on the interaction of the neuronal transmembrane protein Neurexin IV with the glial Ig-domain protein Wrapper. Although Neurexin IV has been previously described to be an essential component of epithelial septate junctions (SJ), we show that its function in mediating glial wrapping at the CNS midline is independent of SJ formation. Moreover, differential splicing generates two different Neurexin IV isoforms. One mRNA is enriched in septate junction-forming tissues, whereas the other mRNA is expressed by neurons and recruited to the midline by Wrapper. Although both Neurexin IV isoforms are able to bind Wrapper, the neuronal isoform has a higher affinity for Wrapper. We conclude that Neurexin IV can mediate different adhesive cell-cell contacts depending on the isoforms expressed and the context of its interaction partners.

Organization and function of the blood-brain barrier in Drosophila.

The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the blood-brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function.

Glial cell migration in the eye disc.

Any complex nervous system is made out of two major cell types, neurons and glial cells. A hallmark of glial cells is their pronounced ability to migrate. En route to their final destinations, glial cells are generally guided by neuronal signals. Here we show that in the developing visual system of Drosophila glial cell migration is largely controlled by glial-glial interactions and occurs independently of axonal contact. Differentiation into wrapping glia is initiated close to the morphogenetic furrow. Using single cell labeling experiments we identified six distinct glial cell types in the eye disc. The migratory glial population is separated from the wrapping glial cells by the so-called carpet cells, extraordinary large glial cells, each covering a surface area of approximately 10,000 epithelial cells. Subsequent cell ablation experiments demonstrate that the carpet glia regulates glial migration in the eye disc epithelium and suggest a new model underlying glial migration and differentiation in the developing visual system.

Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding.

Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect beta-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that beta-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, beta-Spectrin is associated with alpha-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of beta-Spectrin significantly reduces levels of neuronal alpha-Spectrin expression, whereas gain of beta-Spectrin leads to an increase in alpha-Spectrin protein expression. Because the loss of alpha-spectrin does not result in an embryonic nervous system phenotype, beta-Spectrin appears to act at least partially independent of alpha-Spectrin to control axonal patterning.

Development of the peripheral glial cells in Drosophila.

In complex organisms the nervous system comprises two cell types: neurons and glial cells. Their correct interplay is of crucial importance during both the development of the nervous system and for later function of the nervous system. In recent years tools have been developed for Drosophila that enable genetic approaches to understanding glial development and differentiation. Focusing on peripheral glial cells we first summarize wild-type development, then introduce some of the relevant genes that have been identified. Despite obvious differences between Drosophila and mammalian glial cells, the molecular machinery that controls terminal differentiation appears well conserved.

Mummy encodes an UDP-N-acetylglucosamine-dipohosphorylase and is required during Drosophila dorsal closure and nervous system development.

Throughout development cell-cell interactions are of pivotal importance. Cells bind to each other or share information via secreted signaling molecules. To a large degree, these processes are modulated by post-translational modifications of membrane proteins. Glycan-chains are frequently added to membrane proteins and assist their exact function at the cell surface. In addition, the glycosylation pathway is required to generate GPI-linkage in the endoplasmatic reticulum. Here, we describe the analysis of the cabrio/mummy gene, which encodes an UDP-N-acetylglucosamine diphosphorylase. This is a well-conserved and central enzyme in the glycosylation pathway. As expected from this central role in glycosylation, cabrio/mummy mutants show many phenotypic traits ranging from CNS fasciculation defects to defects in dorsal closure and eye development. These phenotypes correlate well with specific glycosylation and GPI-anchorage defects in mummy mutants.

Neuron-glia interaction in the insect nervous system.

In all complex organisms, glial cells are pivotal for neuronal development and function. Insects are characterized by having only a small number of these cells, which nevertheless display a remarkable molecular diversity. An intricate relationship between neurons and glia is initially required for glial migration and during axonal patterning. Recent data suggest that in organisms such as Drosophila, a prime role of glial cells lies in setting boundaries to guide and constrain axonal growth.

The Drosophila cell survival gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj.

The Drosophila gene discs lost (dlt) has been reported to encode a homolog of the vertebrate tight junction PDZ protein Patj, and was thought to play a role in cell polarity. Using rescue experiments and sequence analyses, we show that dlt mutations disrupt the Drosophila Codanin-1 homolog, a cytoplasmic protein, and not the PDZ protein. Mutations in human Codanin-1 are associated with congenital dyserythropoietic anemia type I (CDA I). In Drosophila, the genomic organization of dlt is unusual. dlt shares its first untranslated exon with alpha-spectrin, and both genes are coexpressed throughout development. We show that dlt is not required for cell polarity but is needed for cell survival and cell cycle progression. Finally, we present evidence that the PDZ protein previously thought to be encoded by dlt is not required for viability. We propose to rename this PDZ protein after its vertebrate homolog, Patj (Pals-associated tight junction protein).