Number Of Active Zones Research Articles

A presynaptic role of microtubule-associated protein 1/Futsch in Drosophila: regulation of active zone number and neurotransmitter release.

Structural microtubule-associated proteins (MAPs), like MAP1, not only control the stability of microtubules, but also interact with postsynaptic proteins in the nervous system. Their presynaptic role has barely been studied. To tackle this question, we used the Drosophila model in which there is only one MAP1 homolog: Futsch, which is expressed at the larval neuromuscular junction, presynaptically only. We show that Futsch regulates neurotransmitter release and active zone density. Importantly, we provide evidence that this role of Futsch is not just the consequence of its microtubule-stabilizing function. Using high-resolution microscopy, we show that Futsch and microtubules are almost systematically present in close proximity to active zones, with Futsch being localized in-between microtubules and active zones. Using proximity ligation assays, we further demonstrate the proximity of Futsch, but not microtubules, to active zone components. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying microtubule cytoskeleton.

The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction

Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. Here, we investigate the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compare them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). We find that lethality and locomotor phenotypes correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons.

Activity‐dependent Wnt 7 dendritic targeting in hippocampal neurons: Plasticity‐ and tagging‐related retrograde signaling mechanism?

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Neuron-Type Specific Functions of DNT1, DNT2 and Spz at the Drosophila Neuromuscular Junction

Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional in vivo analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. Here, we investigate the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll 10b, in neurons only, rescued the semi-lethality of spz 2 and DNT1 41 , DNT2 e03444 double mutants, indicating retrograde functions in neurons. In spz 2 mutants, DNT1 41 , DNT2 e03444 double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in spz 2 mutants and DNT1 41 , DNT2 e03444 double mutants, but frequency and amplitude of spontaneous events were reduced in spz 2 mutants suggesting defective neurotransmission. Our data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ.

α-Synuclein A30P decreases neurodegeneration and increases synaptic vesicle release probability in CSPα-null mice

α-Synuclein and Cysteine-string protein-α (CSPα) are presynaptic proteins that participate in the maintenance of synaptic function. Mutations or overexpression of the wild type form of α-synuclein have been related to Parkinson's disease, and CSPα mutations cause one type of neuronal ceroid lipofuscinosis. Both are adult-onset neurodegenerative diseases characterized by neuronal protein aggregations. Strikingly, while in mouse the lack of CSPα produces defective neurotransmission and neurodegeneration of motor terminals, blindness and early lethality, the moderate overexpression of wild-type α-synuclein fully rescues the CSPα-null phenotype. Contrarily, the overexpression of the mutated human α-synuclein A30P (α-synucleinhA30P) has much less effect in CSPα KO mice. To explore how the A30P mutation affects the neuroprotective function of α-synuclein we investigated synaptic structure and neurotransmission in motor nerve terminals of wild-type and CSPα-null mice transgenic for α-synucleinhA30P. We found that although α-synucleinhA30P did not fully prevent neurodegeneration, it significantly improved synaptic organization and function in CSPα-null mice by enhancing quantal content, release probability, synaptic vesicle content, active zone number, postsynaptic area, and microtubule appearance. These results demonstrate that α-synucleinhA30P is able to ameliorate synapse degeneration, despite its apparent lack of functionality and its long-term pathogenic effects in neurons. These findings may help to understand better the dual function of α-synuclein regarding neurodegeneration.This article is part of the Special Issue entitled ‘The Synaptic Basis of Neurodegenerative Disorders’.

DLin-7 Is Required in Postsynaptic Lamina Neurons to Prevent Light-Induced Photoreceptor Degeneration in Drosophila

Inherited retinal degeneration in humans is caused by mutations in a wide spectrum of genes that regulate photoreceptor development and homeostasis. Many of these genes are structurally and functionally conserved in Drosophila, making the fly eye an ideal system in which to study the cellular and molecular basis of blindness. DLin-7, the ortholog of vertebrate MALS/Veli, is a core component of the evolutionarily conserved Crumbs complex. Mutations in any core member of the Crb complex lead to retinal degeneration in Drosophila. Strikingly, mutations in the human ortholog, CRB1, result in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis, two severe retinal dystrophies. Unlike Crumbs, DLin-7 is expressed not only in photoreceptor cells but also in postsynaptic lamina neurons. Here, we show that DLin-7 is required in postsynaptic neurons, but not in photoreceptors such as Crumbs, to prevent light-dependent retinal degeneration. At the photoreceptor synapse, DLin-7 acts as part of a conserved DLin-7/CASK/DlgS97 complex required to control the number of capitate projections and active zones, important specializations in the photoreceptor synapse that are essential for proper neurotransmission. These results are the first to demonstrate that a postsynaptically acting protein prevents light-dependent photoreceptor degeneration and describe a novel, Crumbs-independent mechanism for photoreceptor degeneration.

Passive IR field gradient detection of thermal objects in active Fresnel zones

A dual element Passive-Infrared (PIR) sensor is used with a rotating slit aperture to map a narrow scanning beam on the sensing elements through each lens of a Fresnel lens array. The stimuli generated due to each thermal object fall in the active Fresnel zones in a certain direction based on their locations and temperature variations on the surfaces of the sources. These signals are used to analyze the signatures of stationary thermal objects, their slight movements and thermal field gradient changes of the source surfaces provided the object projected area is less than the area of the active zone. Pattern matching is performed using Dynamic-Time-Warping (DTW) algorithm on STFT reduced length time vectors. The object space is divided into m-active zones that correspond to the Fresnel zones in a Fresnel lens array. Within passive IR region from each of the active zones, the system identifies not only the heat intensity changes but also detects the slight movement of the thermal source. The efficiency of the system is dependent on the number of active Fresnel zones and the angular separation between them. This single node PIR sensor system is designed to cover an angular view of ∼10°×80° while horizontal Field of View (FOV) is divided into 4 active Fresnel zones. Generally costly Thermal-IR camera is used for thermal analysis. Our system is comparatively less costly and active coverage zones are easily configurable by increasing number of Fresnel lenses.

Immunocytochemical identification of synaptotagmin 1, syntaxin 1, Ca2+ channel of the N-type, and nicotinic cholinoreceptor in motor neuromuscular junctions of somatic muscle of the earthworm Lumbricus terrestris

The earthworm somatic muscle contains myoneural synapses forming clusters of “synaptic buttons” in which the proteins syntaxin 1, synaptotagmin 1, and alpha 1B subunit of the Ca2+ channel of the N-type were identified. It is supposed that “synaptic buttons” contain a limited number of active zones, which is due to their small size (1–2 μm) and the pattern of distribution of proteins of the exoendocytotic cycle. The postsynaptic membrane of cholinergic synapses contains nicotinic acetylcholine receptors able to bind alpha-bungarotoxin. The area of the position of receptors on postsynaptic membrane is strongly restricted to the synaptic contact region.

Mutations in DDHD2, Encoding an Intracellular Phospholipase A1, Cause a Recessive Form of Complex Hereditary Spastic Paraplegia

We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease.

Snapin is Critical for Presynaptic Homeostatic Plasticity

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release are largely unknown. We have previously used an electrophysiology-based forward genetic screen to assess the function of >400 neuronally expressed genes for a role in the homeostatic control of synaptic transmission at the neuromuscular junction of Drosophila melanogaster. This screen identified a critical function for dysbindin, a gene linked to schizophrenia in humans (Dickman and Davis, 2009). Biochemical studies in other systems have shown that Snapin interacts with Dysbindin, prompting us to test whether Snapin might be involved in the mechanisms of synaptic homeostasis. Here, we demonstrate that loss of snapin blocks the homeostatic modulation of presynaptic vesicle release following inhibition of postsynaptic glutamate receptors. This is true for both the rapid induction of synaptic homeostasis induced by pharmacological inhibition of postsynaptic glutamate receptors, and the long-term expression of synaptic homeostasis induced by the genetic deletion of the muscle-specific GluRIIA glutamate receptor subunit. Loss of snapin does not alter baseline synaptic transmission, synapse morphology, synapse growth, or the number or density of active zones, indicating that the block of synaptic homeostasis is not a secondary consequence of impaired synapse development. Additional genetic evidence suggests that snapin functions in concert with dysbindin to modulate vesicle release and possibly homeostatic plasticity. Finally, we provide genetic evidence that the interaction of Snapin with SNAP25, a component of the SNARE complex, is also involved in synaptic homeostasis.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high‐pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

Presynaptic active zone density during development and synaptic plasticity

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.

S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.

Interaction between synaptic dynamics and synaptic configuration determines the phase of the response to rhythmic inputs

The postsynaptic response of a neuron to time-varying inputs is determined by the interaction of presynaptic spike times with the short-term dynamics of each synapse. For a neuron driven by stochastic synapses and operating in afluctuation-driven regime, synaptic depression results in a quite different postsynaptic response to a large population input depending on how correlated in time the spikes across individual synapses are [1,2]. Here we show that not only the rate but the phase of the postsynaptic response to a rhythmic population input varies as a function of synaptic dynamics and synaptic configuration, which contributes to input correlations. In computer simulations, a single- compartment spiking model neuron is fed inputs via a synaptic pathway containing M=512 vesicle release sites. The pathway is configured in different ways by assigning release sites equally between varying numbers of active zones (N). Each active zone is driven by a particular input spike train. Spike trains to different active zones have the same time-varying mean frequency but individual spikes are uncorrelated in time. A completely uncorrelated pathway consists of 512 active zones containing a single release site, each driven by an independent presynaptic spike train. In contrast, a completely correlated pathway consists of a single active zone containing 512 releasable vesicles that may all release independently on the arrival of a spike from a single presynaptic cell. Each release site is modelled stochastically: a site may or may not contain a vesicle; an empty release site may be refilled at a fixed rate; a full site may release its vesicle with a given probability on arrival of a presynaptic spike. The refilling rate may be slow compared to the arrival rate of spikes, resulting in synaptic depression. The probability of vesicle release may increase with each spike, decaying back to baseline at a slow rate, resulting in facilitation of release. In the results shown here, the refilling rate is 2/s and the facilitation decay rate is also 2/s. The input signal is a 30Hz carrier frequency that is modulated sinusoidally between 50Hz and 10Hz at different modulation frequencies. This signal is carried by up to 512 independent non-homogeneous Poisson-distributed spike trains. The phase of the output spiking response (obtained from multiple runs with the same and different input trains), relative to the input signal, is plotted against the number of active zones (N) in Figure ​Figure1.1. At all modulation frequencies (0.1, 1, 3Hz) and in all configurations, synaptic depression results in the output response leading the input oscillations. The lead increases as the inputs become more correlated (decreasing N). The effect is amplified by facilitation for correlated synapses (N=1 or 2), but reduced for less correlated pathways. Also, the lead decreases with increasing modulation frequency for correlated synapses, but is maximal at 1Hz for uncorrelated pathways. Figure 1 Phase of response to rhythmic population inputs for different synaptic configurations and modulation frequencies. D: depression; D+F: depression + facilitation.

Calcium Channels Link the Muscle-Derived Synapse Organizer Laminin β2 to Bassoon and CAST/Erc2 to Organize Presynaptic Active Zones

Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC β1b or β4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin β2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin β2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin β2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin β2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.

Neuroligin 2 Is Required for Synapse Development and Function at theDrosophilaNeuromuscular Junction

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.

Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle.

Differences in Ca 2+ regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae

We determined whether two classes of Drosophila larval motor terminals with known differences in structure and transmitter release also showed differences in Ca 2+ regulation. Larval motor neurons can be separated into those producing large synaptic boutons (Ib) and those with small boutons (Is). Ib terminals release less transmitter during single action potentials (APs) than Is terminals, but show greater facilitation during high-frequency stimulation. We measured Ca 2+ transients produced by single APs and AP trains after loading the terminals with the dextran-conjugated Ca 2+ indicator Oregon Green 488 BAPTA-1 (OGB-1). The two pairs of Is and Ib terminals innervating muscle fiber 4 and fibers 6 and 7 were examined. The OGB-1 concentrations were measured in order to compare measurements from terminals with similar OGB-1 loading. For single APs, the change in OGB-1 fluorescence (ΔF/F) in Is boutons was significantly larger than in Ib boutons due to greater Ca 2+ influx per bouton volume. The Is boutons had greater surface area and active zone number per bouton volume than Ib boutons; this could account for the differences in Ca 2+ influx and argues for similar Ca 2+ influx at Is and Ib active zones. As previously reported for the Ib boutons, the distal Is boutons had larger single-AP Ca 2+ transients than proximal ones on muscle fibers 6 and 7, but not on fiber 4. This difference was not due to proximal–distal differences in surface area or active zones per bouton volume and may be due to greater Ca 2+ influx at distal active zones. During AP trains, the Is Ca 2+ transients were larger in amplitude and had longer decay time constants than Ib ones. This can be explained by a slower rate of Ca 2+ extrusion from the Is boutons apparently due to lower plasma membrane Ca 2+ ATPase activity at Is boutons compared to Ib boutons.

A Presynaptic Homeostatic Signaling System Composed of the Eph Receptor, Ephexin, Cdc42, and CaV2.1 Calcium Channels

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release remain largely unknown. In a screen, we isolated mutations in Drosophila ephexin (Rho-type guanine nucleotide exchange factor) that disrupt the homeostatic enhancement of presynaptic release following impairment of postsynaptic glutamate receptor function at the Drosophila neuromuscular junction. We show that Ephexin is sufficient presynaptically for synaptic homeostasis and localizes in puncta throughout the nerve terminal. However, ephexin mutations do not alter other aspects of neuromuscular development, including morphology or active zone number. We then show that, during synaptic homeostasis, Ephexin functions primarily with Cdc42 in a signaling system that converges upon the presynaptic CaV2.1 calcium channel. Finally, we show that Ephexin binds the Drosophila Eph receptor (Eph) and Eph mutants disrupt synaptic homeostasis. Based on these data, we propose that Ephexin/Cdc42 couples synaptic Eph signaling to the modulation of presynaptic CaV2.1 channels during the homeostatic enhancement of presynaptic release.