Moesin helps to restrain synaptic growth at the Drosophila neuromuscular junction

The precise role of actin and actin-binding proteins in synaptic development is unclear. In Drosophila, overexpression of a dominant-negative NSF2 construct perturbs filamentous actin, which is associated with overgrowth of the NMJ, while co-expression of moesin, which encodes an actin binding protein, suppresses this overgrowth phenotype. These data suggest that Moesin may play a role in synaptic development at the Drosophila NMJ. To further investigate this possibility, we examined the influence of loss-of-function moesin alleles on the NSF2-induced overgrowth phenotype. We found that flies carrying P-element insertions that reduce moesin expression enhanced the NMJ overgrowth phenotype, indicating a role for Moesin in normal NMJ morphology. In addition to the NMJ overgrowth phenotype, expression of dominant-negative NSF2 is known to reduce the frequency of miniature excitatory junctional potentials and the amplitude of excitatory junctional potentials. We found that moesin coexpression did not restore the physiology of the mutant NSF2 phenotype. Together, our results demonstrate a role for moesin in regulating synaptic growth in the Drosophila NMJ and suggest that the effect of dominant-negative NSF2 on NMJ morphology and physiology may have different underlying molecular origins.     

SSB, an antigen that selectively labels morphologically distinct synaptic boutons at the Drosophila larval neuromuscular junction.

In this report we describe the expression of Small Synaptic Bouton (SSB), an antigen that is selectively expressed in a specific subset of neuromuscular junction terminals in the body wall of Drosophila larva. The expression of SSB was studied with a polyclonal antibody raised against the cAMP phosphodiesterase of the Drosophila learning mutant dunce (Nighorn et al., 1991, Neuron 6:455-467); however, immunoreactivity was not abolished by the dunce (dnc) alleles dncM14 and dncM11 or deficiencies of the dnc gene, indicating that the antigen labelled could not be the dnc gene product, but another antigen that we termed SSB. Immunoreactivity was localized in the body wall muscles to a specific subset of neuromuscular junction terminals that have been implicated in activity-dependent plasticity. This demonstrates that these morphologically distinct terminals can be immunocytochemically distinguished and that they probably represent innervation by a distinct neuronal population. Confocal and electron microscopic examination demonstrated that staining was restricted to the synaptic boutons themselves, not to neurites or motor axons. Ultrastructural analysis showed label close to synaptic vesicles in the presynaptic terminal and in the surrounding subsynaptic reticulum. Central nervous system (CNS) staining was restricted to a segmentally repeated pattern of cell bodies in the ventral ganglion and to a few small groups of cells in the brain lobes.     

The Drosophila miR-310 cluster negatively regulates synaptic strength at the neuromuscular junction

Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.     

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.     

Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction

The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity‐dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools. We provide In vivo evidence at the single gene level that the calcium‐dependent adenylyl cyclase rutabaga , the phosphodiesterase dunce , the kinase PKA, and Protein Phosphatase 1 (PP1) all operate within a functional signaling pathway to modulate Sema2a‐dependent chemorepulsion. It was found that presynaptic cAMP levels were required to be dynamically maintained at an optimal level to suppress connectivity defects. It was also proposed that PP1 may serve as a molecular link between cAMP signaling and CaMKII in the pathway underlying refinement. The results introduced an in vivo model where presynaptic cAMP levels, downstream of electrical activity and calcium influx, act via PKA and PP1 to modulate the neuron's response to chemorepulsion involved in the withdrawal of off‐target synaptic contacts. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 39–60, 2017     

The pharmacological and physiological profile of glutamate receptors at the Drosophila larval neuromuscular junction

Abstract.  Drosophila larval muscles are commonly used for developmental assessment in regard to various mutations of synaptically relevant molecules. In addition, the molecular sequence of the glutamate receptors on the muscle fibre have been described; however, the pharmacological profiles to known agonists and antagonists have yet to be reported. Here, the responses of N -methyl- d -aspartic acid, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), l -glutamate, kainate, quisqualic acid, NBQX, AP5 and DNQX are characterized with regard to synaptic transmission and direct effects on the muscle fibres. The muscle fibres depolarize to application of glutamate or quisqualate and the excitatory postsynaptic potential (EPSP) amplitudes are diminished. Kainate does not alter the muscle membrane potential but does reduce the EPSP amplitude. The known antagonists NBQX, AP5 and DNQX have no substantial effect on synaptic transmission at 1 m m , nor do they block the response of quisqualate. Kainate may be acting as a postsynaptic antagonist or via autoreceptors presynaptically to reduce evoked transmission.     

A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila

A family of three position-specific (PS) integrins are expressed at the Drosophila neuromuscular junction (NMJ): a beta subunit ((betaPS), expressed in both presynaptic and postsynaptic membranes, and two alpha subunits (alphaPS1, alphaPS2), expressed at least in the postsynaptic membrane. PS integrins appear at postembryonic NMJs coincident with the onset of rapid morphological growth and terminal type-specific differentiation, and are restricted to type I synaptic boutons, which mediate fast, excitatory glutamatergic transmission. We show that two distinctive hypomorphic mutant alleles of the beta subunit gene myospheroid (mys(b9) and mys(ts1)), differentially affect betaPS protein expression at the synapse to produce distinctive alterations in NMJ branching, bouton formation, synaptic architecture and the specificity of synapse formation on target cells. The mys(b9) mutation alters betaPS localization to cause a striking reduction in NMJ branching, bouton size/number and the formation of aberrant 'mini-boutons', which may represent a developmentally arrested state. The mys(ts1) mutation strongly reduces betaPS expression to cause the opposite phenotype of excessive synaptic sprouting and morphological growth. NMJ function in these mutant conditions is altered in line with the severity of the morphological aberrations. Consistent with these mutant phenotypes, transgenic overexpression of the betaPS protein with a heat-shock construct or tissue-specific GAL4 drivers causes a reduction in synaptic branching and bouton number. We conclude that betaPS integrin at the postembryonic NMJ is a critical determinant of morphological growth and synaptic specificity. These data provide the first genetic evidence for a functional role of integrins at the postembryonic synapse.     

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.     

TOR is required for the retrograde regulation of synaptic homeostasis at the Drosophila neuromuscular junction

Homeostatic mechanisms operate to stabilize synaptic function; however, we know little about how they are regulated. Exploiting Drosophila genetics, we have uncovered a critical role for the target of rapamycin (TOR) in the regulation of synaptic homeostasis at the Drosophila larval neuromuscular junction. Loss of postsynaptic TOR disrupts a retrograde compensatory enhancement in neurotransmitter release that is normally triggered by a reduction in postsynaptic glutamate receptor activity. Moreover, postsynaptic overexpression of TOR or a phosphomimetic form of S6 ribosomal protein kinase, a common target of TOR, can trigger a strong retrograde increase in neurotransmitter release. Interestingly, heterozygosity for eIF4E, a critical component of the cap-binding protein complex, blocks the retrograde signal in all these cases. Our findings suggest that cap-dependent translation under the control of TOR plays a critical role in establishing the activity dependent homeostatic response at the NMJ.     

Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction

Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se , adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron‐dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Barfly: sculpting membranes at the Drosophila neuromuscular junction

The ability of a cell to change the shape of its membranes is intrinsic to many cellular functions. Proteins that can alter or recognize curved membrane structures and those that can act to recruit other proteins which stabilize the membrane curvature are likely to be essential in cell functions. The BAR (Bin, amphiphysin, RVS167 homology) domain is a protein domain that can either induce lipidic membranes to curve or can sense curved membranes. BAR domains are found in several proteins at neuronal synapses. We will review BAR domain structure and the role that BAR domain containing proteins play in regulating the morphology and function of the Drosophila neuromuscular junction. In flies the BAR domain containing proteins, endophilin and syndapin affect synaptic vesicle endocytosis, whereas CIP4, dRich, nervous wreck and syndapin affect synaptic morphology. We will review the growing evidence implicating mutations in BAR domain containing proteins being the cause of human pathologies.