Glial processes at the Drosophila larval neuromuscular junction match synaptic growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。     

Glutamatergic synapses of Drosophila neuromuscular junctions: a high-resolution model for the analysis of experience-dependent potentiation

The glutamatergic synapses of developing neuromuscular junctions (NMJ) of Drosophila larvae are readily accessible, morphologically simple, and physiologically well-characterized. They therefore have a long and highly successful tradition as a model system for the discovery of genetic and molecular mechanisms of target recognition, synaptogenesis, NMJ development, and synaptic plasticity. However, since the development and the activity-dependent refinement of NMJs are concurrent processes, they cannot easily be separated by the widely applied genetic manipulations that mostly have chronic effects. Recent studies have therefore begun systematically to incorporate larval foraging behavior into the physiological and genetic analysis of NMJ function in order to analyze potential experience-dependent changes of glutamatergic transmission. These studies have revealed that recent crawling experience is a potent modulator of glutamatergic transmission at NMJs, because high crawling activities result after an initial lag-phase in several subsequent phases of experience-dependent synaptic potentiation. Depending on the time window of occurrence, four distinct phases of experience-dependent potentiation have been defined. These phases of potentiation can be followed from their initial induction (phase-I) up to the morphological consolidation (phase-III/IV) of previously established functional changes (phase-II). This therefore establishes, for the first time, a temporal hierarchy of mechanisms involved in the use-dependent modification of glutamatergic synapses.     

GDNF acutely potentiates Ca2+ channels and excitatory synaptic transmission in midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) is best known for its long-term survival effect on dopaminergic neurons in the ventral midbrain. A recent study showed that acute application of GDNF to these neurons suppresses A-type potassium channels and potentiates neuronal excitability. Here we have characterized the acute effects of GDNF on Ca(2+) channels and synaptic transmission. GDNF rapidly and reversibly potentiated the high voltage-activated (HVA) Ca(2+) channel currents in cultured dopaminergic neurons. Analyses of channel kinetics indicate that GDNF decreased the activation time constant, increased the inactivation and deactivation time constants of HVA Ca(2+) channel currents. Ca(2+) imaging experiments demonstrate that GDNF facilitated Ca(2+) influx induced by membrane depolarization. To investigate the physiological consequences of the Ca(2+) channel modulation, we examined the acute effects of GDNF on excitatory synaptic transmission at synapses made by these dopaminergic neurons, which co-release the transmitter glutamate. Within 3 min of application, GDNF increased the amplitude of spontaneous and evoked excitatory autaptic- or multiple-postsynaptic currents. The frequency as well as the amplitude of miniature excitatory postsynaptic currents was also increased. These results reveal, for the first time, an acute effect of GDNF on synaptic transmission and its potential mechanisms, and suggest that an important function of GDNF for midbrain dopaminergic neurons is the acute modulation of transmission and ion channels.     

Neurotrophic regulation of the development and function of the neuromuscular synapses

Recent studies have established that one of the major functions of neurotrophic factors is to regulate synaptic development and plasticity. This owes a great deal to the studies using the neuromuscular junction (NMJ) as a model system. In this review, we summarize the effects of various neurotrophic factors on the development and function of the neuromuscular synapses. We describe experiments addressing the role of neurotrophins, as well as that of other factors (GFLs, TGF-βs, and Wnts). The synaptic effects of neurotrophic factors are divided into two categories: acute effects on synaptic transmission and plasticity occurring within seconds or minutes after cells are exposed to a particular factor, and long-term regulation of synaptic structure and function that takes days to accomplish. We consider the presynaptic effects on the release of the neurotransmitter ACh, as well as the postsynaptic effects on the clustering of ACh receptors. Further studies of the mechanisms underlying these regulatory effects will help us better understand how neurotrophic factors can achieve diverse and synapse-specific modulation in the brain.     

Prodomain Removal Enables Neto to Stabilize Glutamate Receptors at the Drosophila Neuromuscular Junction

Stabilization of neurotransmitter receptors at postsynaptic specializations is a key step in the assembly of functional synapses. Drosophila Neto (Neuropillin and Tolloid-like protein) is an essential auxiliary subunit of ionotropic glutamate receptor (iGluR) complexes required for the iGluRs clustering at the neuromuscular junction (NMJ). Here we show that optimal levels of Neto are crucial for stabilization of iGluRs at synaptic sites and proper NMJ development. Genetic manipulations of Neto levels shifted iGluRs distribution to extrajunctional locations. Perturbations in Neto levels also produced small NMJs with reduced synaptic transmission, but only Neto-depleted NMJs showed diminished postsynaptic components. Drosophila Neto contains an inhibitory prodomain that is processed by Furin1-mediated limited proteolysis. neto null mutants rescued with a Neto variant that cannot be processed have severely impaired NMJs and reduced iGluRs synaptic clusters. Unprocessed Neto retains the ability to engage iGluRs in vivo and to form complexes with normal synaptic transmission. However, Neto prodomain must be removed to enable iGluRs synaptic stabilization and proper postsynaptic differentiation.     

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance

Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.     

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.     

GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.     

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.     

Basal lamina and the organization of neuromuscular synapses

Fast chemical synapses are comprised of presynaptic and postsynaptic specializations precisely aligned across a protein-filled synaptic cleft. At the vertebrate neuromuscular junction (NMJ), the synaptic cleft contains a structured form of extracellular matrix known as a basal lamina (BL). Synaptic BL is molecularly differentiated from the BL that covers the extrasynaptic region of the myofiber. This review summarizes current understanding of the morphology, composition, and function of the synaptic BL at the vertebrate NMJ. Considerable evidence supports the conclusion that the synaptic BL organizes and maintains pre- and postsynaptic specializations during development and regeneration, and promotes robust neurotransmission in the adult.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor β (TGFβ). We have provided evidence that TGFβ regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRα1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFβ, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFβ. Additional treatment with TGFβ does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRα2 and GFRα1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRα1, TGFβ is not required for recruitment of GFRα2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFβ. Disruption of TGFβ signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRα1 crystallization and ontogenetic development of the CG that may explain the differences in TGFβ-dependence of GDNF and NRTN.     

Neurturin, a member of the glial cell line-derived neurotrophic factor family, affects the development of acetylcholinesterase-positive cells in a three-dimensional model system of retinogenesis

The glial cell line-derived neurotrophic factor (GDNF) family consists of the four ligands GDNF, neurturin (NRTN), artemin and persephin, which bind to the four co-receptors GDNF family receptor alpha1-4 and control through the activation of the receptor tyrosin kinase Ret several developmental processes. The purpose of this study was to analyse the expression and the influence of NRTN in the developing retina. We used retinospheres, a three-dimensional model system of the developing chicken retina. The expression of NRTN and the GDNF family receptor alpha 2 increased during development. Furthermore, expression was comparable in retinae and retinospheres. Analysis of signalling pathways influenced by NRTN in retinospheres showed activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinase (MAPK). Activation of MAPK could be localised in cells of the innermost rows of the inner nuclear layer which were predominantly acetylcholinesterase-positive cells. Exogenous application of NRTN increased the amount of acetylcholinesterase-positive cells within the retinospheres at late culture stages. Additionally, we could show that Müller glia cells did not express the GFRalpha2 receptor and were probably not involved in NRTN signalling. Therefore, we conclude that NRTN directly participates in regulatory processes concerning the differentiation of acetylcholinesterase-positive cells in the chicken retina.     

Ca(2+) binding protein frequenin mediates GDNF-induced potentiation of Ca(2+) channels and transmitter release

Molecular mechanisms underlying long-term neurotrophic regulation of synaptic transmission and plasticity are unknown. We report here that long-term treatment of neuromuscular synapses with glial cell line-derived neurotrophic factor (GDNF) potentiates spontaneous and evoked transmitter release, in ways very similar to presynaptic expression of the Ca(2+) binding protein frequenin. GDNF enhances the expression of frequenin in motoneurons, and inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca(2+) influx into the nerve terminals during evoked transmission by enhancing Ca(2+) currents. The effect of GDNF on Ca(2+) currents is blocked by inhibition of frequenin expression, occluded by overexpression of frequenin, and is selective to N-type Ca(2+) channels. These results identify an important molecular target that mediates the long-term, synaptic action of a neurotrophic factor.     

Investigating the safety factor at an invertebrate neuromuscular junction

Fidelity of synaptic transmission is essential at the neuromuscular junction (NMJ). To ensure that transmission does not fail, vertebrate motoneurons often release more neurotransmitter than is required for muscle contraction. This safety factor allows some loss of synaptic function without failure of muscle contraction. It is not known whether a similar mechanism operates at the invertebrate neuromuscular junction. In our study of the Drosophila NMJ, we find that glutamate receptor mutants can exhibit a substantial decrease in synaptic function while maintaining muscle contraction. The persistence of neuromuscular function in these mutants is not explained by synaptic facilitation, temporal summation of high frequency stimuli, or a hyperpolarizing shift in the activation range of muscle calcium channels. Instead, the attenuated synaptic response is sufficient to drive muscle contraction. Quantitative analysis of the decrease in synaptic transmission in these mutants implies that at the wild-type NMJ there is an approximately five- to ninefold excess in released transmitter. Hence, the presence of a synaptic safety factor is a conserved feature of neuromuscular organization in both invertebrates and vertebrates.