Building a synapse

L-glutamate is the primary neurotransmitter at excitatory synapses in the vertebrate CNS and at arthropod neuromuscular junctions (NMJs). However, the molecular mechanisms that trigger the recruitment of glutamate receptors at the onset of synaptogenesis and promote their stabilization at postsynaptic densities remain poorly understood. We have reported the discovery of a novel, evolutionary conserved molecule, Neto, essential for clustering of ionotropic glutamate receptors (iGluRs) at Drosophila NMJ. Neto is the first auxiliary subunit described in Drosophila and is the only non-channel subunit absolutely required for functional iGluRs. Here we review the role of Drosophila Neto in synapse assembly, its similarities with other Neto proteins and a new perspective on how glutamatergic synapses are physically assembled and stabilized.     

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.     

Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.     

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.     

The adult abdominal neuromuscular junction of Drosophila: a model for synaptic plasticity

During its life cycle, Drosophila makes two sets of neuromuscular junctions (NMJs), embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval NMJs are restructured to give rise to their adult counterparts, a process that is integrated into the overall remodeling of the nervous system. The NMJs of the prothoracic muscles and the mesothoracic dorsal longitudinal (flight) muscles have been previously described. Given the diversity and complexity of adult muscle groups, we set out to examine the less complex abdominal muscles. The large bouton sizes of these NMJs are particularly advantageous for easy visualization. Specifically, we have characterized morphological attributes of the ventral abdominal NMJ and show that an embryonic motor neuron identity gene, dHb9, is expressed at these adult junctions. We quantified bouton numbers and size and examined the localization of synaptic markers. We have also examined the formation of boutons during metamorphosis and examined the localization of presynaptic markers at these stages. To test the usefulness of the ventral abdominal NMJs as a model system, we characterized the effects of altering electrical activity and the levels of the cell adhesion molecule, FasciclinII (FasII). We show that both manipulations affect NMJ formation and that the effects are specific as they can be rescued genetically. Our results indicate that both activity and FasII affect development at the adult abdominal NMJ in ways that are distinct from their larval and adult thoracic counterparts     

wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.     

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

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.     

Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction

L-glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+-dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L-glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies.     

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。     

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.     

Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila

Here we incorporate recent advances in Drosophila neurogenetics and "optogenetics" into neuroscience laboratory exercises. We used the light-activated ion channel channelrhodopsin-2 (ChR2) and tissue-specific genetic expression techniques to study the neural basis of behavior in Drosophila larvae. We designed and implemented exercises using inexpensive, easy-to-use systems for delivering blue light pulses with fine temporal control. Students first examined the behavioral effects of activating glutamatergic neurons in Drosophila larvae and then recorded excitatory junctional potentials (EJPs) mediated by ChR2 activation at the larval neuromuscular junction (NMJ). Comparison of electrically and light-evoked EJPs demonstrates that the amplitudes and time courses of light-evoked EJPs are not significantly different from those generated by electrical nerve stimulation. These exercises introduce students to new genetic technology for remotely manipulating neural activity, and they simplify the process of recording EJPs at the Drosophila larval NMJ. Relatively little research work has been done using ChR2 in Drosophila, so students have opportunities to test novel hypotheses and make tangible contributions to the scientific record. Qualitative and quantitative assessment of student experiences suggest that these exercises help convey principles of synaptic transmission while also promoting integrative and inquiry-based studies of genetics, cellular physiology, and animal behavior.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Visualization of neuropeptide expression,transport, and exocytosis in Drosophila melanogaster

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP‐tagged atrial natriuretic factor fusion (preproANF‐EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF‐EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF‐EMD was concentrated in nerve terminals. We compared the release of proANF‐EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate‐containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46‐fold more neuropeptide and released much more proANF‐EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 159–172, 2001     

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.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

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.     

latheo, a Drosophila gene involved in learning, regulates functional synaptic plasticity.

Mutations in the latheo (lat) gene disrupt associative learning in Drosophila , but a role for LAT in regulating neuronal function has not been demonstrated. Here, we report that LAT plays a central role in regulating Ca2(+)- and activity-dependent synaptic plasticity. Immunological localization of the LAT protein indicates it is present at synaptic connections of the larval neuromuscular junction (NMJ) and is enriched in presynaptic boutons. Basal synaptic transmission amplitude at the lat mutant NMJ is elevated 3- to 4-fold, and Ca2+ dependence of transmission is significantly reduced. Multiple forms of synaptic facilitation and posttetanic potentiation (PTP) are strongly depressed or absent at the mutant synapse. Our results suggest that LAT is a novel presynaptic protein with a role in the Ca2(+)-dependent synaptic modulation mechanisms necessary for behavioral plasticity.     

Direct influence of serotonin on the larval heart of Drosophila melanogaster

The heart rate (HR) of larval Drosophila is established to be modulated by various neuromodulators. Serotonin (5-HT) showed dose-dependent responses in direct application within semi-intact preparations. At 1 nM, HR decreased by 20% while it increased at 10 nM (10%) and 100 nM (30%). The effects plateaued at 100 nM. The action of 5-HT on the heart was examined with an intact Central Nervous System (CNS) and an ablated CNS. The heart and aorta of dorsal vessel pulsate at different rates at rest and during exposure to 5-HT. Splitting the heart and aorta resulted in a dramatic reduction in pulse rate of both the segments and the addition of 5-HT did not produce regional differences. The split aorta and heart showed a high degree of sensitivity to sham changes of saline but no significant effect to 5-HT. Larvae-fed 5-HT (1 mM) did not show any significant change in HR. Since 3,4-methylenedioxymethamphetamine (MDMA) is known to act as a weak agonist on 5-HT receptors in vertebrates, we tested an exogenous application; however, no significant effect was observed to dosage ranging from 1 nM to 100 μM in larvae with and without an intact CNS. In summary, direct application of 5-HT to the larval heart had significant effects in a dose-dependent manner while MDMA had no effect.