A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.
Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled “Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster” from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.
Drosophila melanogaster; Giant Fiber Circuit; screening; in vivo; nanoinjection; electrophysiology; modulatory compounds
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 however 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-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 (hiw). 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 central nervous system.
giant fiber; Drosophila; synapse formation; ubiquitin; growth; maturation
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR’s ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.
Experiments in peripheral and central synapses reveal the regulatory mechanisms that enable trans-synaptic control of synapse development and maintenance by the L1-type CAM Neuroglian.
The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg–Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.
The function of neuronal circuits relies on precise connectivity, and processes like learning and memory involve refining this connectivity through the selective formation and elimination of synapses. Cell adhesion molecules (CAMs) that directly mediate cell–cell interactions at synaptic contacts are thought to mediate this structural synaptic plasticity. In this study, we used an unbiased genetic screen to identify the Drosophila L1-type CAM Neuroglian as a central regulator of synapse formation and maintenance. We show that the intracellular Ankyrin interaction motif, which links Neuroglian to the cytoskeleton, is an essential regulatory site for Neuroglian mobility, adhesion, and synaptic function. In motoneurons, the strength of Ankyrin binding directly controls the balance between synapse formation and maintenance. At a central synapse, however, a dynamic regulation of the Neuroglian–Ankyrin interaction is required to coordinate transsynaptic development. Our study identifies the interaction of the L1-type CAM with Ankyrin as a novel regulatory module enabling local and precise control of synaptic connectivity without altering general neuronal circuit architecture. This interaction is relevant for normal nervous system development and disease as mutations in L1-type CAMs cause mental retardation and psychiatric diseases in humans.
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.
Neuromodulation; conotoxins; ImI; in vivo drug screening assay; acetylcholine receptor antagonists; neuronal circuit function; electrophysiology; Drosophila
The Drosophila standard brain has been a useful tool that provides information about position and size of different brain structures within a wild-type brain and allows the comparison of imaging data that were collected from individual preparations. Therefore the standard can be used to reveal and visualize differences of brain regions between wild-type and mutant brains and can provide spatial description of single neurons within the nervous system. Recently the standard brain was complemented by the generation of a ventral nerve cord (VNC) standard. Here the authors have registered the major components of a simple neuronal circuit, the Giant Fiber System (GFS), into this standard. The authors show that they can also virtually reconstruct the well-characterized synaptic contact of the Giant Fiber with its motorneuronal target when they register the individual neurons from different preparations into the VNC standard. In addition to the potential application for the standard thorax in neuronal circuit reconstruction, the authors show that it is a useful tool for in-depth analysis of mutant morphology of single neurons. The authors find quantitative and qualitative differences when they compared the Giant Fibers of two different neuroglian alleles, nrg849 and nrgG00305, using the averaged wild-type GFS in the standard VNC as a reference.
Giant Fiber; neuronal circuit; Neuroglian; Drosophila; standard brain; ventral nerve cord
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.
We observed that neither muscle development nor animal longevity was compromised in these transgenic animals. However, human APP expressing adults developed age-dependent defects in both climbing and flying. We could advance or retard the onset of symptoms by rearing animals in vials with different surface properties, suggesting that human APP expression-mediated behavioral defects are influenced by muscle activity. Muscles from transgenic animals did not display protein aggregates or structural abnormalities at the light or transmission electron microscopic levels. In agreement with genetic studies performed with developing mammalian myoblasts, we observed that co-expression of the ubiquitin E3 ligase Parkin could ameliorate human APP-induced defects.
These data suggest that: 1) ectopic expression of human APP in fruit flies leads to age- and activity-dependent behavioral defects without overt changes to muscle development or structure; 2) environmental influences can greatly alter the phenotypic consequences of human APP toxicity; and 3) genetic modifiers of APP-induced pathology can be identified and analyzed in this model.
amyloid precursor protein (APP); Drosophila; muscle; mitochondria; electron microscopy; apoptosis; Parkin
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 article 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 giant fiber system.
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.
Giant fiber; axon pathfinding; synaptogenesis; L1-CAM; Drosophila; cell adhesion
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.
Cell adhesion; Ankyrins; Membrane skeleton; Tyrosine phosphorylation; Neurite outgrowth; Neuromuscular junction; Synapse; Synaptogenesis; Drosophila
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.
giant fiber; Drosophila; synapse formation; ubiquitin; growth; maturation