ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo evenness interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell-autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

ESCRT disruption provides evidence against transsynaptic signaling functions for extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a "ceiling effect," because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.

Recording from Drosophila Larval Body Wall Muscles: Passive Membrane Properties and Basic Features of Synaptic Transmission.

The Drosophila larval body wall muscle preparation was first used for electrophysiological analysis in the 1970s. This preparation has become the "gold standard" for studying neuronal excitability as well as synaptic transmission. Here, we first describe the steps for performing intracellular recording from fly larval body wall muscles and then explain how to record and analyze spontaneous and evoked synaptic potentials. Methods used include larval dissection (filleting), identification of muscle fibers and their innervating nerves, the use of the micromanipulator and microelectrode in penetrating the muscle membrane, and nerve stimulation to evoke synaptic potentials.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.

Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.

Electrophysiological Recording from a "Model" Cell.

The muscle cell or neuron membrane is functionally equivalent to a resistor-capacitor (RC) circuit with the membrane resistance and capacitor in parallel. Once inserted inside the membrane, an electrode introduces a serial resistance and small capacitance to the RC circuit. Through a narrow opening at its tip (∼0.1-µm), current can pass through the electrode, into the cell, and back to the outside (ground) across the membrane to complete the circuit. This arrangement enables a voltage difference between the outside and inside of the cell membrane to be recorded. To determine cell membrane properties, a current can be injected into the cell through the electrode. One complication with this approach, however, is that the voltage difference measured with the electrode includes the voltage drop across the cell membrane and that across the electrode. Furthermore, a small amount of current is drawn by the electrode capacitor, thereby slowing the current flow across the membrane. Fortunately, most amplifiers are equipped with bridge balance and capacitance compensation functions so that the effects of the electrode on cell membrane properties can be canceled out or minimized. This protocol describes the basics of setting up and conducting electrophysiological experiments using a model cell. For the novice, a model cell provides a way to learn the operation of electrophysiology equipment and software without the anxiety of damaging living cells. This protocol also illustrates passive membrane properties such as the input resistance, capacitance, and time constant.

Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording in Drosophila.

Electrophysiological recording is a group of techniques used to record electrical field potentials generated by cells. These techniques rely on several types of electrodes, which can be manufactured in the laboratory. In intracellular recording, glass microelectrodes are used to pierce the cell membrane, and then to measure the electrical potential difference between the inside and the outside of the cell. Another technique, called loose patch or focal recording, is similar to intracellular recording but the electrode tip does not pierce into the cell membrane. Rather, the electrode tip is placed near a nerve or the postsynaptic side of the neuromuscular junction (NMJ) to record extracellular changes in local potentials. A third technique involves a suction electrode, which is used to draw part of the motor nerve into the electrode so that electrical pulses can be applied to elicit action potentials of the nerve. Suction electrodes are specifically used to evoke synaptic potentials at the Drosophila larval NMJ. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.

Synaptic Electrophysiology of the Drosophila Neuromuscular Junction.

Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal experiences an influx of calcium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neurotransmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SVs at presynaptic terminals. The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic features of synaptic transmission, as well as the molecular mechanisms regulating the SV cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ is an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Here, we describe the theory and practice of electrophysiology as applied to the Drosophila larval NMJ preparation. We introduce the basics of membrane potentials, with an emphasis on the resting potential and synaptic potential. We also describe the equipment and methods required to set up an electrophysiology rig.

Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues.

3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples, or from pluripotent stem cells, can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity, and the appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD-, and iPSC-derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery.

Neuronal extracellular vesicles (EVs) are locally released from presynaptic terminals, carrying cargoes critical for intercellular signaling and disease. EVs are derived from endosomes, but it is unknown how these cargoes are directed to the EV pathway rather than for conventional endolysosomal degradation. Here, we find that endocytic machinery plays an unexpected role in maintaining a release-competent pool of EV cargoes at synapses. Endocytic mutants, including nervous wreck (nwk), shibire/dynamin, and AP-2, unexpectedly exhibit local presynaptic depletion specifically of EV cargoes. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo synaptotagmin-4 (Syt4) and suppress lethality upon overexpression of the EV cargo amyloid precursor protein (APP). These EV defects are genetically separable from canonical endocytic functions in synaptic vesicle recycling and synaptic growth. Endocytic machinery opposes the endosomal retromer complex to regulate EV cargo levels and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that locally promotes cargo loading into synaptic EVs.

Electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues.

Recently, methods for creating three-dimensional (3-D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study, we provide an electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues, focusing on Duchenne muscular dystrophy (DMD). We enlist the 3-D in vitro model of DMD muscle tissue to evaluate muscle cell electrical properties uncoupled from presynaptic neural inputs, an understudied aspect of DMD. Our data show that previously reported electrophysiological aspects of DMD, including effects on membrane potential and membrane resistance, are replicated in the 3-D muscle tissue model. Furthermore, we test a potential therapeutic compound, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle. Therefore, this study serves as a baseline for a new in vitro method to examine potential therapies for muscular disorders.

Disruption of Drosophila larval muscle structure and function by UNC45 knockdown.

BACKGROUND: Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. MAIN RESULTS: UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. CONCLUSIONS: This study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle.

The biological basis of Duchenne muscular dystrophy (DMD) pathology is only partially characterized and there are still few disease-modifying therapies available, therein underlying the value of strategies to model and study DMD. Dystrophin, the causative gene of DMD, is responsible for linking the cytoskeleton of muscle fibers to the extracellular matrix beyond the sarcolemma. We posited that disease-associated phenotypes not yet captured by two-dimensional culture methods would arise by generating multinucleated muscle cells within a three-dimensional (3D) extracellular matrix environment. Herein we report methods to produce 3D human skeletal muscle microtissues (hMMTs) using clonal, immortalized myoblast lines established from healthy and DMD donors. We also established protocols to evaluate immortalized hMMT self-organization and myotube maturation, as well as calcium handling, force generation, membrane stability (i.e., creatine kinase activity and Evans blue dye permeability) and contractile apparatus organization following electrical-stimulation. In examining hMMTs generated with a cell line wherein the dystrophin gene possessed a duplication of exon 2, we observed rare dystrophin-positive myotubes, which were not seen in 2D cultures. Further, we show that treating DMD hMMTs with a ß1-integrin activating antibody, improves contractile apparatus maturation and stability. Hence, immortalized myoblast-derived DMD hMMTs offer a pre-clinical system with which to investigate the potential of duplicated exon skipping strategies and those that protect muscle cells from contraction-induced injury. STATEMENT OF SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder that is caused by mutation of the dystrophin gene. The biological basis of DMD pathology is only partially characterized and there is no cure for this fatal disease. Here we report a method to produce 3D human skeletal muscle microtissues (hMMTs) using immortalized human DMD and healthy myoblasts. Morphological and functional assessment revealed DMD-associated pathophysiology including impaired calcium handling and de novo formation of dystrophin-positive revertant muscle cells in immortalized DMD hMMTs harbouring an exon 2 duplication, a feature of many DMD patients that has not been recapitulated in culture prior to this report. We further demonstrate that this "DMD in a dish" system can be used as a pre-clinical assay to test a putative DMD therapeutic and study the mechanism of action.

Dual- and single-shot susceptibility ratio measurements with circular polarizations in second-harmonic generation microscopy.

Polarization-resolved second-harmonic generation (P-SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio χzzz2'/χzxx2' , with z-axis parallel and x-axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P-SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of χzzz2'/χzxx2' : A dual-shot configuration where the SHG circular anisotropy generated using incident right- and left-handed circularly-polarized light is measured; and a single-shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the χzzz2'/χzxx2' of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes-Mueller polarimetry. The dual- and single-shot circular anisotropy measurements can be used for fast imaging that is independent of the in-plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.

The neuronal ceroid lipofuscinosis protein Cln7 functions in the postsynaptic cell to regulate synapse development.

The neuronal ceroid lipofuscinoses (NCLs) are a group of fatal, monogenic neurodegenerative disorders with an early onset in infancy or childhood. Despite identification of the genes disrupted in each form of the disease, their normal cellular role and how their deficits lead to disease pathology is not fully understood. Cln7, a major facilitator superfamily domain-containing protein, is affected in a late infantile-onset form of NCL. Cln7 is conserved across species suggesting a common function. Here we demonstrate that Cln7 is required for the normal growth of synapses at the Drosophila larval neuromuscular junction. In a Cln7 mutant, synapses fail to develop fully leading to reduced function and behavioral changes with dysregulation of TOR activity. Cln7 expression is restricted to the post-synaptic cell and the protein localizes to vesicles immediately adjacent to the post-synaptic membrane. Our data suggest an involvement for Cln7 in regulating trans-synaptic communication necessary for normal synapse development.

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres.

Analysis of Drosophila nervous system development following an early, brief exposure to ethanol.

The effects of ethanol on neural function and development have been studied extensively, motivated in part by the addictive properties of alcohol and the neurodevelopmental deficits that arise in children with fetal alcohol spectrum disorder (FASD). Absent from this research area is a genetically tractable system to study the effects of early ethanol exposure on later neurodevelopmental and behavioral phenotypes. Here, we used embryos of the fruit fly, Drosophila melanogaster, as a model system to investigate the neuronal defects that arise after an early exposure to ethanol. We found several disruptions of neural development and morphology following a brief ethanol exposure during embryogenesis and subsequent changes in larval behavior. Altogether, this study establishes a new system to examine the effects of alcohol exposure in embryos and the potential to conduct large-scale genetics screens to uncover novel factors that sensitize or protect neurons to the effects of alcohol.

A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction.

Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

Imaging SNAP-29 in Drosophila.

SNAP-29 is expressed throughout the life cycle of fruit fly and exhibits wide tissue distribution patterns. Unlike other SNAP-25-like proteins (i.e., SNAP-25, SNAP-23/24, and SNAP-47) which primarily support exocytosis at the plasma membrane, SNAP-29 regulates various intracellular trafficking events, by partnering with proteins active in both exocytosis and endocytosis. Here we describe the protocol to localize SNAP-29 in early embryos, imaginal discs from third instar larva, and immortalized S2 cells via immunofluorescence microscopy.

Regulation of SH3PX1 by dNedd4-long at the Drosophila neuromuscular junction.

Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic-specific regulation of SH3PX1 by dNedd4Lo.