Drosophila Central Nervous System Research Articles

Copper overload and deficiency both adversely affect the central nervous system of Drosophila.

The human copper homeostasis disorders Menkes and Wilson disease both have severe neurological symptoms. Menkes is a copper deficiency disorder whereas Wilson disease patients suffer from copper toxicity, indicating that tight control of neuronal copper levels is essential for proper nervous system development and function. Here we examine the consequences of neuronal copper deficiency and excess in the Drosophila melanogaster nervous system, using targeted manipulation of the copper uptake genes Ctr1A and Ctr1B and efflux gene ATP7 in combination with altered dietary copper levels. We find that pan-neuronal over expression of Ctr1B and ATP7 both result in a reduction in viability. The effects of Ctr1B over expression are exacerbated by dietary copper supplementation and rescued by copper limitation indicating a copper toxicity phenotype. Dietary manipulation has the opposite effect on ATP7 over expression, indicating that this causes neuronal copper deficiency due to excessive copper efflux. Copper deficiency also causes a highly penetrant developmental defect in surviving adult flies which can be replicated by both copper excess and copper deficiency targeted specifically to a small subset of neuropeptidergic cells. We conclude that both copper overload and excess have detrimental effects on Drosophila neuronal function, reducing overall fly viability as well as impacting on a specific neuropeptide pathway.

Origin and specification of the brain leucokinergic neurons of Drosophila: Similarities to and differences from abdominal leucokinergic neurons

The Drosophila central nervous system contains many types of neurons that are derived from a limited number of progenitors as evidenced in the ventral ganglion. The situation is much more complex in the developing brain. The main neuronal structures in the adult brain are generated in the larval neurogenesis, although the basic neuropil structures are already laid down during embryogenesis. The embryonic factors involved in adult neuron origin are largely unknown. To shed light on how brain cell diversity is achieved, we studied the early temporal and spatial cues involved in the specification of lateral horn leucokinin peptidergic neurons (LHLKs). Our analysis revealed that these neurons have an embryonic origin. We identified their progenitor neuroblast as Pcd6 in the Technau and Urbach terminology. Evidence was obtained that a temporal series involving the transcription factors Kr, Pdm, and Cas participates in the genesis of the LHLK lineage, the Castor window being the one in which the LHLKs neurons are generated. It was also shown that Notch signalling and Dimmed are involved in the specification of the LHLKs. Serial homologies with the origin and factors involved in specification of the abdominal leucokinergic neurons (ABLKs) have been detected.

Live Imaging of Neuroblast Lineages within Intact Larval Brains in Drosophila

Neuroblasts are the precursors of the Drosophila central nervous system and undergo repeated physical and molecular asymmetric cell divisions. Live imaging of neuroblast lineages within intact Drosophila larval brains has dramatically improved our current understanding of basic cellular processes such as the establishment of cell polarity, spindle orientation, and cytokinesis. The analysis of mutant phenotypes using live imaging can enlarge our understanding of asymmetric neuroblast division and self-renewal. Although much live neuroblast imaging is performed using green fluorescent protein only, the generation of improved fluorescent proteins has led to an increase in the use of two-color imaging. Here we present a simple protocol for isolating and imaging larval brain neuroblasts. We describe procedures for the dissection and mounting of brains from third-instar Drosophila larvae in explant solution and their subsequent live imaging. The method provides a close approximation to the in vivo environment and produces data with high temporal and spatial resolutions. We also discuss potential problems and pitfalls and provide examples of how this technique is used.

5′-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the Drosophila central nervous system

Drosophila RNP-4F is a highly conserved protein from yeast to human and functions as a spliceosome assembly factor during pre-mRNA splicing. Two major developmentally regulated rnp-4f mRNA isoforms have been described during fly development, designated “long” and “short,” differing by a 177-nt tract in the 5′-UTR. This region potentially folds into a single long stable stem–loop by pairing of intron 0 and part of exon 2. Since the coding potential for the two isoforms is identical, the interesting question arises as to the functional significance of this evolutionarily-conserved 5′-UTR feature. Here we describe the effects of wild-type and mutated stem–loop on modulation of rnp-4f gene expression in embryos using a GFP reporter assay. In this work, a new GFP expression vector designated pUAS-Neostinger was constructed. The UAS-GAL4 system was utilized to trigger GFP expression using tissue-specific promoter driver fly lines. Fluorescence microscopy visualization, Western blotting and real-time qRT-PÇR were used to study and quantify GFP reporter protein and mRNA levels. A significant increase in GFP reporter protein expression due to presence of the wild-type stem–loop sequence/structure was unexpectedly observed with no concomitant increase in GFP reporter mRNA levels, showing that the 177-nt region enhancement acts posttranscriptionally. The effects of potential cis-acting elements within the stem–loop were evaluated using the reporter assay in two mutant constructs. Results of GFP reporter over-expression show that RNP-4F translational regulation is highly sensitive in the developing fly central nervous system. The potential molecular mechanism behind the observed translational enhancement is discussed.

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system

Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim–Tgo binding sites whereas only 1 Sim–Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim–Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim–Tgo binding sites were added. Taken together, these results suggest that the number of Sim–Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes.

Commissureless Regulation of Axon Outgrowth across the Midline Is Independent of Rab Function

Nervous system function requires that neurons within neural circuits are connected together precisely. These connections form during the process of axon guidance whereby each neuron extends an axon that migrates, often large distances, through a complex environment to reach its synaptic target. This task can be simplified by utilising intermediate targets to divide the route into smaller sections. This requires that axons adapt their behaviour as they migrate towards and away from intermediate targets. In the central nervous system the midline acts as an intermediate target for commissural axons. In Drosophila commissural axons switch from attraction towards to extension away from the midline by regulating the levels of the Roundabout receptor on their cell surface. This is achieved by Commissureless which directs Roundabout to an intracellular compartment in the soma prior to reaching the midline. Once across the midline Roundabout is allowed to reach the surface and acts as a receptor for the repellent ligand Slit that is secreted by cells at the midline. Here we investigated candidate intracellular mechanisms that may facilitate the intracellular targeting of Commissureless and Roundabout within the soma of commissural neurons. Using modified forms of Commissureless or Rabs we show that neither ubiquitination nor Rab activity are necessary for the intracellular targeting of Commissureless. In addition we reveal that axon outgrowth of many populations of neurons within the Drosophila central nervous system is also independent of Rab activity.

Mutual inhibition among postmitotic neurons regulates robustness of brain wiring in Drosophila

Brain connectivity maps display a delicate balance between individual variation and stereotypy, suggesting the existence of dedicated mechanisms that simultaneously permit and limit individual variation. We show that during the development of the Drosophila central nervous system, mutual inhibition among groups of neighboring postmitotic neurons during development regulates the robustness of axon target choice in a nondeterministic neuronal circuit. Specifically, neighboring postmitotic neurons communicate through Notch signaling during axonal targeting, to ensure balanced alternative axon target choices without a corresponding change in cell fate. Loss of Notch in postmitotic neurons modulates an axon's target choice. However, because neighboring axons respond by choosing the complementary target, the stereotyped connectivity pattern is preserved. In contrast, loss of Notch in clones of neighboring postmitotic neurons results in erroneous coinnervation by multiple axons. Our observations establish mutual inhibition of axonal target choice as a robustness mechanism for brain wiring and unveil a novel cell fate independent function for canonical Notch signaling. DOI:http://dx.doi.org/10.7554/eLife.00337.001.

Whole-Cell In Vivo Patch-Clamp Recordings in the Drosophila Brain

Whole-cell patch-clamp recordings provide exceptional access to spiking and synaptic neural activity. This method has been applied to neurons in the central nervous system of Drosophila and allows researchers the opportunity to study the function of their neurons of interest within the context of native circuits in a genetically tractable model system. In this protocol, we describe the technique for in vivo whole-cell patch-clamp recordings in a preparation which exposes neurons in the fly brain. We also offer technical suggestions and discuss some of the challenges encountered in recording from single neurons in the fly brain. Neurons are patched following routine recording protocols for whole-cell patch clamp. At the physiology rig, additional cleaning of the brain is performed to allow easy access to the neurons, and the cells can be filled with a diffusible dye during recordings, to examine the morphology of the recorded cell post hoc. In the electrophysiology rig used for Drosophila patch-clamp recordings, the microscope stage has been removed, so that the recording platform instead rests on a ring stand support that is magnetically fixed to the table. Manipulators and stimulus delivery are also in fixed locations, whereas the microscope sits on an x-y translation stage.

Dissection of the Head Cuticle and Sheath of Living Flies for Whole-Cell Patch-Clamp Recordings in the Brain

Whole-cell patch-clamp recording has been applied to neurons in the central nervous system of Drosophila, allowing researchers to study the function of neurons of interest within native circuits in a genetically tractable model system. Here, we present a method to expose neurons in the fly brain for such recording. The fly is inserted into an opening in a piece of tinfoil in a recording platform and fixed in place. This immobilizes the fly's head and permits the exposed neurons to be bathed in saline above the surface of the foil, while the sensory structures (antennae, proboscis, and much of the eyes) remain dry below the surface of the foil. The tinfoil is within a well that allows external saline to be perfused over the fly, and the walls of the well adapted so the ground wire (connected to the amplifier head stage) can be securely positioned within the saline bath. Our method to construct a recording platform, presented here, makes use of materials in the laboratory. Once the fly is mounted in the platform, the head cuticle and sheath covering the brain are removed. We describe how to mount the fly and open up the head cuticle to access the mushroom body Kenyon cells (KCs), whose somata are located on the dorsal/posterior surface of the brain. To record from neuronal cell bodies located in other brain regions, the approach to mount the fly must be modified to provide consistent access to the cells of interest.

A Resource for Manipulating Gene Expression and Analyzing cis-Regulatory Modules in the Drosophila CNS

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data setsare available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizinginterneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

Deterministic or Stochastic Choices in Retinal Neuron Specification

There are two views on vertebrate retinogenesis: a deterministic model dependent on fixed lineages and a stochastic model in which choices of division modes and cell fates cannot be predicted. In this issue of Neuron, He etal. (2012) address this question in zebrafish using live imaging and mathematical modeling.

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

SummaryNeuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

Comparison of Parallel High-Throughput RNA Sequencing Between Knockout of TDP-43 and Its Overexpression Reveals Primarily Nonreciprocal and Nonoverlapping Gene Expression Changes in the Central Nervous System of Drosophila

The human Tar-DNA binding protein, TDP-43, is associated with amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. TDP-43 contains two conserved RNA-binding motifs and has documented roles in RNA metabolism, including pre-mRNA splicing and repression of transcription. Here, using Drosophila melanogaster as a model, we generated loss-of-function and overexpression genotypes of Tar-DNA binding protein homolog (TBPH) to study their effect on the transcriptome of the central nervous system (CNS). By using massively parallel sequencing methods (RNA-seq) to profile the CNS, we find that loss of TBPH results in widespread gene activation and altered splicing, much of which are reversed by rescue of TBPH expression. Conversely, TBPH overexpression results in decreased gene expression. Although previous studies implicated both absence and mis-expression of TDP-43 in ALS, our data exhibit little overlap in the gene expression between them, suggesting that the bulk of genes affected by TBPH loss-of-function and overexpression are different. In combination with computational approaches to identify likely TBPH targets and orthologs of previously identified vertebrate TDP-43 targets, we provide a comprehensive analysis of enriched gene ontologies. Our data suggest that TDP-43 plays a role in synaptic transmission, synaptic release, and endocytosis. We also uncovered a potential novel regulation of the Wnt and BMP pathways, many of whose targets appear to be conserved.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

Alumina nanoparticles alter rhythmic activities of local interneurons in the antennal lobe of Drosophila

The special physical and chemical properties of nanomaterials open up new capabilities and functions. However, concerns have been raised about the risks produced by nanoparticles, their potential to cause undesirable effects, such as contamination of the environment and other adverse effects. In this study, we used Drosophila as a model organism to explore the effects of nano-alumina on the central nervous system. We focused on the rhythmic activities in the antennal lobe of Drosophila using patch clamps to record the electrophysiological activities. We found that 15 min after application of alumina nanoparticles, the average frequencies of spontaneous activities were significantly decreased compared with control groups (0.65 ± 0.13 Hz, 0.34 ± 0.07 Hz, *p < 0.05). These results indicated that nano-alumina might have adverse effects on the central nervous system in Drosophila.

Drosophila TRPA Channel Painless Inhibits Male–Male Courtship Behavior through Modulating Olfactory Sensation

The Drosophila melanogaster TRPA family member painless, expressed in a subset of multidendritic neurons embeding in the larval epidermis, is necessary for larval nociception of noxious heat or mechanical stimuli. However, the function of painless in adult flies remains largely unknown. Here we report that mutation of painless leads to a defect in male–male courtship behavior and alteration in olfaction sensitivity in adult flies. Specific downregulation of the expression of the Painless protein in the olfactory projection neurons (PNs) of the antennal lobes (ALs) resulted in a phenotype resembling that found in painless mutant flies, whereas overexpression of Painless in PNs of painless mutant males suppressed male–male courtship behavior. The downregulation of Painless exclusively during adulthood also resulted in male–male courtship behavior. In addition, mutation of the painless gene in flies caused changes in olfaction, suggesting a role for this gene in olfactory processing. These results indicate that functions of painless in the adult central nervous system of Drosophila include modulation of olfactory processing and inhibition of male–male courtship behavior.

Dissection of Third-Instar Drosophila Larvae for Electrophysiological Recording from Neurons

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. It is possible to obtain electrophysiological recordings from all stages of Drosophila. Exposure of the intact Drosophila CNS is a prerequisite for such electrophysiological recordings. The dissection procedure described here is suitable for third-instar larvae. The dissection should take ∼5 min to complete if all preparation work has been completed in advance. Owing to the short life span of the dissected larva, it is not recommended that the procedure be stopped or the preparation stored for later use.

Dissection of First- and Second-Instar Drosophila Larvae for Electrophysiological Recording from Neurons: The Flat (or Fillet) Preparation: Figure 1.

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. It is possible to obtain electrophysiological recordings from all stages of Drosophila. Exposure of the intact Drosophila CNS is a prerequisite for such electrophysiological recordings. The dissection procedure described here can be applied to both late-stage embryos (stage 16 onward) and larvae. Because of their size, third-instar larvae are more difficult to flatten using this method and, if recording from this stage, the reader might consider using insect pins for the dissection or isolating the CNS using an alternative method. The dissection should take <10 min if all preparation work has been completed in advance. Owing to the short life span of the dissected larva, it is not recommended that the procedure be stopped or the preparation stored for later use.

Heparan sulfate proteoglycan specificity during axon pathway formation in the Drosophila embryo

Axon guidance is influenced by the presence of heparan sulfate (HS) proteoglycans (HSPGs) on the surface of axons and growth cones (Hu, [2001]: Nat Neurosci 4:695-701; Irie et al. [2002]: Development 129:61-70; Inatani et al. [2003]: Science 302:1044-1046; Johnson et al. [2004]: Curr Biol 14:499-504; Steigemann et al. [2004]: Curr Biol 14:225-230). Multiple HSPGs, including Syndecans, Glypicans and Perlecans, carry the same carbohydrate polymer backbones, raising the question of how these molecules display functional specificity during nervous system development. Here we use the Drosophila central nervous system (CNS) as a model to compare the impact of eliminating Syndecan (Sdc) and/or the Glypican Dally-like (Dlp). We show that Dlp and Sdc share a role in promoting accurate patterns of axon fasciculation in the lateral longitudinal neuropil; however, unlike mutations in sdc, which disrupt the ability of the secreted repellent Slit to prevent inappropriate passage of axons across the midline, mutations in dlp show neither midline defects nor genetic interactions with Slit and its Roundabout (Robo) receptors at the midline. Dlp mutants do show genetic interactions with Slit and Robo in lateral fascicle formation. In addition, simultaneous loss of Dlp and Sdc demonstrates an important role for Dlp in midline repulsion, reminiscent of the functional overlap between Robo receptors. A comparison of HSPG distribution reveals a pattern that leaves midline proximal axons with relatively little Dlp. Finally, the loss of Dlp alters Slit distribution distal but not proximal to the midline, suggesting that distinct yet overlapping pattern of HSPG expression provides a spatial system that regulates axon guidance decisions.

Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ40 and Aβ42, the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions.