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In JoVE (2)
- Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection
- Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System
Other Publications (13)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Neuron
- Nature Neuroscience
- Development (Cambridge, England)
- The European Journal of Neuroscience
- Seminars in Cell & Developmental Biology
- Current Biology : CB
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Cellular & Molecular Biology Letters
- Journal of Neurogenetics
- Cold Spring Harbor Protocols
- Toxicon : Official Journal of the International Society on Toxinology
- BMC Physiology
Articles by Tanja A. Godenschwege in JoVE
JoVE Neuroscience
Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection
Department of Biological Sciences, Florida Atlantic University
An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.
JoVE Neuroscience
Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System
1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.
Other articles by Tanja A. Godenschwege on PubMed
Ectopic Expression in the Giant Fiber System of Drosophila Reveals Distinct Roles for Roundabout (Robo), Robo2, and Robo3 in Dendritic Guidance and Synaptic Connectivity
The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.
New Roles for Ubiquitin in the Assembly and Function of Neuronal Circuits
A series of recent papers highlight a prominent role for ubiquitin in the formation and function of neural circuits. These new results focus attention on the molecular remodeling that occurs at various decision points in the life of growth cones and synapses.
Bi-directional Signaling by Semaphorin 1a During Central Synapse Formation in Drosophila
Semaphorins have been intensively studied for their role in dendritic and axonal pathfinding, but less is known about their potential role in synapse formation. In the adult giant fiber (GF) system of fruit flies (Drosophila melanogaster), we show that transmembrane Semaphorin 1a (Sema-1a) is involved in synapse formation in addition to its role in guidance during pathfinding. Cell-autonomous rescue experiments showed that Sema-1a is involved in assembly of a central synapse and that it is required both pre- and postsynaptically. We also found that pre- but not postsynaptic gain-of-function Sema-1a was able to disrupt the GF-motor neuron synapse and that the phenotype depended on a proline-rich intracellular domain that contains a putative Enabled binding site. We suggest that transmembrane Sema-1a is part of a bi-directional signaling system that leads to the formation of the GF synapse and possibly acts as both a ligand and a receptor.
Targeted Expression of Shibire Ts and Semaphorin 1a Reveals Critical Periods for Synapse Formation in the Giant Fiber of Drosophila
In order to determine the timing of events during the assembly of a neural circuit in Drosophila we targeted expression of the temperature-sensitive shibire gene to the giant fiber system and then disrupted endocytosis at various times during development. The giant fiber retracted its axon or incipient synapses when endocytosis was blocked at critical times, and we perceived four phases to giant fiber development: an early pathfinding phase, an intermediate phase of synaptogenesis, a late stabilization process and, finally, a mature synapse. By co-expressing shibire(ts) and semaphorin 1a we provided evidence that Semaphorin 1a was one of the proteins being regulated by endocytosis and its removal was a necessary part of the program for synaptogenesis. Temporal control of targeted expression of the semaphorin 1a gene showed that acute excess Semaphorin 1a had a permanent disruptive effect on synapse formation.
Flies Lacking All Synapsins Are Unexpectedly Healthy but Are Impaired in Complex Behaviour
Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.
Making an Escape: Development and Function of the Drosophila Giant Fibre System
Flies escape danger by jumping into the air and flying away. The giant fibre system (GFS) is the neural circuit that mediates this simple behavioural response to visual stimuli. The sensory signal is received by the giant fibre and relayed to the leg and wing muscle motorneurons. Many of the neurons in the Drosophila GFS are uniquely identifiable and amenable to cell biological, electrophysiological and genetic studies. Here we review the anatomy and development of this system and highlight its utility for studying many aspects of nervous system biology ranging from neural development and synaptic plasticity to the aetiology of neural disorder.
A Conserved Role for Drosophila Neuroglian and Human L1-CAM in Central-synapse Formation
Drosophila Neuroglian (Nrg) and its vertebrate homolog L1-CAM are cell-adhesion molecules (CAM) that have been well studied in early developmental processes. Mutations in the human gene result in a broad spectrum of phenotypes (the CRASH-syndrome) that include devastating neurological disorders such as spasticity and mental retardation. Although the role of L1-CAMs in neurite extension and axon pathfinding has been extensively studied, much less is known about their role in synapse formation.
A Mechanism Distinct from Highwire for the Drosophila Ubiquitin Conjugase Bendless in Synaptic Growth and Maturation
The signaling mechanisms that allow the conversion of a growth cone into a mature and stable synapse are yet to be completely understood. Ubiquitination plays key regulatory roles in synaptic development and may be involved in this process. Previous studies identified the Drosophila ubiquitin conjugase bendless (ben) to be important for central synapse formation, but the precise role it plays has not been elucidated. Our studies indicate that Ben plays a pivotal role in synaptic growth and maturation. We have determined that an incipient synapse is present with a high penetrance in ben mutants, suggesting that Ben is required for a developmental step after target recognition. We used cell-autonomous rescue experiments to show that Ben has a presynaptic role in synapse growth. We then harnessed the TARGET system to transiently express UAS (upstream activating sequence)-ben in a ben mutant background and identified a well defined critical period for Ben function in establishing a full-grown, mature synaptic terminal. We demonstrate that the protein must be present at a time point before but not during the actual growth process. We also provide phenotypic evidence demonstrating that Ben is not a part of the signal transduction pathway involving the well characterized ubiquitin ligase highwire. We conclude that Bendless functions as a novel developmental switch that permits the transition from axonal growth and incipient synapse formation to synaptic growth and maturation in the CNS.
The Interaction Between L1-type Proteins and Ankyrins--a Master Switch for L1-type CAM Function
L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that ankyrin-binding to L1-type CAMs provides a master switch. The interaction with ankyrins directs L1-type adhesive proteins into different functional contexts, either ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into ankyrin-dependent functions, such as L1's role at axon initial segments (AIS), paranodal regions, synapses and in dendrites.
Genetic Interaction of Neuroglian and Semaphorin1a During Guidance and Synapse Formation
We have previously demonstrated a function for Neuroglian and Semaphorin1a in Drosophila giant fiber circuit formation. Both molecules are required for guiding the giant fibers out of the brain and have distinct functions during giant synapse formation. In this study we characterized the effects of various combinations of Neuroglian and Semaphorin1a gain and loss of function backgrounds on giant fiber circuitry formation. We found that Neuroglian and Semaphorin1a genetically interact with each other during axon guidance as well as during synapse formation. Our experiments revealed that during pathfinding of the giant fibers out of the brain, Neuroglian function seems to be dependent on Semaphorin1a. In contrast, during giant fiber synapse formation we observed that Semaphorin1a signaling as a receptor can be altered by Neuroglian in the same cell. In summary, our findings suggest that Neuroglian and Semaphorin1a can regulate each other's function in cis and that the resultant signaling output is possibly different during guidance and synapse formation.
Electrophysiological Recordings from the Drosophila Giant Fiber System (GFS)
INTRODUCTION: The giant fiber system (GFS) of Drosophila is a well-characterized neuronal circuit that mediates the escape response in the fly. It is one of the few adult neural circuits from which electrophysiological recordings can be made routinely. This protocol describes a simple procedure for stimulating the giant fiber neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS: the tergotrochanteral "jump" muscle (TTM) and the large indirect flight muscles (dorsal longitudinal muscles, or DLMs). It is a relatively noninvasive method that allows the investigator to stimulate the giant fibers in the brain and assay the function of several central synapses within this neural circuit by recording from the thoracic musculature.
A Novel Approach for in Vivo Screening of Toxins Using the Drosophila Giant Fiber Circuit
Finding compounds that affect neuronal or muscular function is of great interest as potential therapeutic agents for a variety of neurological disorders. Alternative applications for these compounds include their use as molecular probes as well as insecticides. We have developed a bioassay that requires small amounts of compounds and allows for unbiased screening of biological activity in vivo. For this, we paired administering compounds in a non-invasive manner with simultaneous electrophysiological recordings from a well-characterized neuronal circuit, the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. The circuit encompasses a variety of neurons with cholinergic, glutamatergic, and electrical synapses as well as neuromuscular junctions. Electrophysiological recordings from this system allow for the detection of compound-related effects against any molecular target on these components. Here, we provide evidence that this novel bioassay works with small molecules such as the cholinergic receptor blocker mecamylamine hydrochloride and the potassium channel blocker tetraethylammonium hydroxide, as well as with venom from Conus brunneus and isolated conopeptides. Conopeptides have been developed into powerful drugs, such as the painkillers Prialt™ and Xen2174. However, most conopeptides have yet to be characterized, revealing the need for a rapid and straightforward screening method. Our findings show that mecamylamine hydrochloride, as well as the α-conotoxin ImI, which is known to be an antagonist of the human α7 nicotinic acetylcholine receptor, efficiently disrupted the synaptic transmission of a Drosophila α7 nicotinic acetylcholine receptor-dependent pathway in our circuit but did not affect the function of neurons with other types of synapses. This demonstrates that our bioassay is a valid tool for screening for compounds relevant to human health.
Expression of Human Amyloid Precursor Protein in the Skeletal Muscles of Drosophila Results in Age- and Activity-dependent Muscle Weakness
One of the hallmarks of Alzheimer's disease, and several other degenerative disorders such as Inclusion Body Myositis, is the abnormal accumulation of amyloid precursor protein (APP) and its proteolytic amyloid peptides. To better understand the pathological consequences of inappropriate APP expression on developing tissues, we generated transgenic flies that express wild-type human APP in the skeletal muscles, and then performed anatomical, electrophysiological, and behavioral analysis of the adults.
