Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.     

Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Synaptic plasticity, neuronal activity‐dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive‐feedback signaling pathways by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca²⁺]_i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive‐feedback reactions, CaMKII autophosphorylation and CaMKII‐mediated inhibition of a CaM‐dependent phosphodiesterase, PDE1. The simulation predicted that PDE1‐mediated feedforward inhibition of CaMKII predominantly controls the Ca²⁺ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca²⁺ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1.     

Recent advances in using Drosophila to model neurodegenerative diseases

Neurodegenerative diseases are progressive disorders of the nervous system that affect the function and maintenance of specific neuronal populations. Most disease cases are sporadic with no known cause. The identification of genes associated with familial cases of these diseases has enabled the development of animal models to study disease mechanisms. The model organism Drosophila has been successfully used to study pathogenic mechanisms of a wide range of neurodegenerative diseases. Recent genetic studies in the Drosophila models have provided new insights into disease mechanisms, emphasizing the roles played by mitochondrial dynamics, RNA (including miRNA) function, protein translation, and synaptic plasticity and differentiation. It is anticipated that Drosophila models will further our understanding of mechanisms of neurodegeneration and facilitate the development of novel and rational treatments for these debilitating neurodegenerative diseases.     

Transmission,Development, and Plasticity of Synapses

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

Synapse maturation and structural plasticity at Drosophila neuromuscular junctions

The Drosophila larval neuromuscular junction has recently emerged as a powerful model system to characterize the cellular and molecular events involved in the formation and flexibility of synapses. The combination of molecular, genetic, electrophysiological and anatomical approaches has revealed, for example, the functional significance of the discs-large gene product (a novel synapse-organizing protein) in the nervous system. This protein is involved in the clustering of at least one ion channel and in the structural modification of glutamatergic synapses during target muscle growth. The manipulation of the genes encoding ion channels, components of second-messenger cascades, and cell adhesion molecules is beginning to tease apart the mechanisms underlying structural synaptic plasticity.     

果蝇产卵行为的偏好和可塑性实验模型

我们用经典的Sherrington模型构建了一个以食物环境为输入,果蝇产卵行为为输出的神经行为学模型,并且对这个模型的敏感性以及可塑性进行了探索性研究．通过给果蝇提供不同的食物成分和浓度,我们发现,果蝇在不同浓度的食物上产卵量有明显偏好差异,并表现出很好的敏感性和稳定性．而当阻断了果蝇的嗅觉感受神经元后,产卵的浓度偏好受到了影响．另一方面,我们用经典条件化的学习方式对果蝇的产卵行为进行训练后,发现果蝇也可以学会将光照条件和产卵行为偶联起来．这也证明了这个模型具有很好的可塑性．这个模型将为今后进一步研究果蝇食物偏好、产卵行为、条件化学习等的神经和分子机制奠定良好的基础．     

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.     

STDP in a bistable synapse model based on CaMKII and associated signaling pathways

The calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in the induction of long-term postsynaptic modifications following calcium entry. Experiments suggest that these long-term synaptic changes are all-or-none switch-like events between discrete states. The biochemical network involving CaMKII and its regulating protein signaling cascade has been hypothesized to durably maintain the evoked synaptic state in the form of a bistable switch. However, it is still unclear whether experimental LTP/LTD protocols lead to corresponding transitions between the two states in realistic models of such a network. We present a detailed biochemical model of the CaMKII autophosphorylation and the protein signaling cascade governing the CaMKII dephosphorylation. As previously shown, two stable states of the CaMKII phosphorylation level exist at resting intracellular calcium concentration, and high calcium transients can switch the system from the weakly phosphorylated (DOWN) to the highly phosphorylated (UP) state of the CaMKII (similar to a LTP event). We show here that increased CaMKII dephosphorylation activity at intermediate Ca²⁺ concentrations can lead to switching from the UP to the DOWN state (similar to a LTD event). This can be achieved if protein phosphatase activity promoting CaMKII dephosphorylation activates at lower Ca²⁺ levels than kinase activity. Finally, it is shown that the CaMKII system can qualitatively reproduce results of plasticity outcomes in response to spike-timing dependent plasticity (STDP) and presynaptic stimulation protocols. This shows that the CaMKII protein network can account for both induction, through LTP/LTD-like transitions, and storage, due to its bistability, of synaptic changes.     

14-3-3 proteins in neuronal development and function

The 14-3-3 proteins are small, cytosolic, evolutionaritly conserved proteins expressed abundantly in the nervous system. Although they were discovered more than 30 yr ago, their function in the nervous system has remained enigmatic. Several recent studies have helped to clarify their biological function. Crystallographic investigations have revealed that 14-3-3 proteins exist as dimers and that they contain a specific region for binding to other proteins. The interacting proteins, in turn, contain a 14-3-3 binding motif; proteins that interact with 14-3-3 dimers include PKC and Raf, protein kinases with critical roles in neuronal signaling. These proteins are capable of activating Raf in vitro, and this role has been verified by in vivo studies inDrosophila. Most interestingly, mutations in theDrosophila 14-3-3 genes disrupt neuronal differentiation, synaptic plasticity, and behavioral plasticity, establishing a role for these proteins in the development and function of the nervous system.     

Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. Here we report that KN-93, a membrane-permeable CaMKII inhibitor, blocked glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocked synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produced an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons.     

NMDA receptors on zebrafish Mauthner cells require CaMKII‐α for normal development

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real‐time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M‐cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII‐α was expressed in the M‐cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII‐α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τ_fast ≈ 30 ms and τ_slow ≈ 300 ms. Knockdown of CaMKII‐α specifically reduced the amplitude of the slow component of the NMDA‐mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M‐cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII‐α altered locomotor behaviors of touch responses. These results suggest that CaMKII‐α is present in embryonic M‐cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M‐cell development. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 145–162, 2015     

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.     

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.     

Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons

Activity-dependent synaptic plasticity is known to be important in learning and memory, persistent pain and drug addiction. Glutamate NMDA receptor activation stimulates several protein kinases, which then trigger biochemical cascades that lead to modifications in synaptic efficacy. Genetic and pharmacological techniques have been used to show a role for Ca²⁺/calmodulin-dependent kinase II (CaMKII) in synaptic plasticity and memory formation. However, it is not known if increasing CaMKII activity in forebrain areas affects behavioral responses to tissue injury. Using genetic and pharmacological techniques, we were able to temporally and spatially restrict the over expression of CaMKII in forebrain areas. Here we show that genetic overexpression of CaMKII in the mouse forebrain selectively inhibits tissue injury-induced behavioral sensitization, including allodynia and hyperalgesia, while behavioral responses to acute noxious stimuli remain intact. CaMKII overexpression also inhibited synaptic depression induced by a prolonged repetitive stimulation in the ACC, suggesting an important role for CaMKII in the regulation of cingulate neurons. Our results suggest that neuronal CaMKII activity in the forebrain plays a role in persistent pain.     

A diffusion-activation model of CaMKII translocation waves in dendrites

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is a key regulator of glutamatergic synapses and plays an essential role in many forms of synaptic plasticity. It has recently been observed that stimulating dendrites locally with a single glutamate/glycine puff induces a local translocation of CaMKII into spines that subsequently spreads in a wave-like manner towards the distal dendritic arbor. Here we present a mathematical model of the diffusion, activation and translocation of dendritic CaMKII. We show how the nonlinear dynamics of CaMKII diffusion-activation generates a propagating translocation wave, provided that the rate of activation is sufficiently fast. We also derive an explicit formula for the wave speed as a function of physiological parameters such as the diffusivity of CaMKII and the density of spines. Our model provides a quantitative framework for understanding the spread of CaMKII translocation and its possible role in heterosynaptic plasticity.     

A comparison of the neuronal dysfunction caused by <Emphasis Type="Italic">Drosophila</Emphasis> tau and human tau in a <Emphasis Type="Italic">Drosophila</Emphasis> model of tauopathies

Hyperphosphorylation and aggregation of tau into tangles is a feature of disorders such as Alzheimer’s disease and other Tauopathies. To model these disorders in Drosophila melanogaster, human tau has been over-expressed and a variety of phenotypes have been observed including neurotoxicity, disrupted neuronal and synaptic function and locomotor impairments. Neuronal dysfunction has been seen prior to neuronal death and in the absence of tangle formation. The Drosophila tau protein shares a large degree of homology with human tau but differs in the crucial microtubule binding domains. Although like human tau Drosophila tau can induce neurotoxicity, little is known about its ability to disrupt neuronal function. In this study we demonstrate that like human tau, over-expression of Drosophila tau results in disrupted axonal transport, altered neuromuscular junction morphology and locomotor impairments. This indicates that like human tau, over-expression of Drosophila tau compromises neuronal function despite significant differences in microtubule binding regions.     

Anatomy, physiology and pharmacology of Caenorhabditis elegans pharynx: a model to define gene function in a simple neural system

Invertebrate neuroscience has provided a number of very informative model systems that have been extensively utilized in order to define the neurobiological bases of animal behaviours (Sattelle and Buckingham in Invert Neurosci 6:1–3, 2006). Most eminent among these are a number of molluscs, including Aplysia californica, Lymnaea stagnalis and Helix aspersa, crustacean systems such as the crab stomatogastric ganglion and a wide-range of other arthropods. All of these have been elegantly exploited to shed light on the very important phenomenon of the molecular and cellular basis for synaptic regulation that underpins behavioural plasticity. Key to the successful use of these systems has been the ability to study well-defined, relatively simple neuronal circuits that direct and regulate a quantifiable animal behaviour. Here we describe the pharyngeal system of the nematode C. elegans and its utility as a model for defining the genetic basis of behaviour. The circuitry of the nervous system in this animal is uniquely well-defined. Furthermore, the feeding behaviour of the worm is controlled by the activity of the pharynx and this in turn is regulated in a context-dependent manner by a simple nervous system that integrates external signals, e.g. presence or absence of food, and internal signals, e.g. the nutritional status of the animal to direct an appropriate response. The genetics of C. elegans is being effectively exploited to provide novel insight into genes that function to regulate the neuronal network that controls the pharynx. Here we summarise the progress to date and highlight topics for future research. Two main themes emerge. First, although the anatomy of the pharyngeal system is very well-defined, there is a much poorer understanding of its neurochemistry. Second, it is evident that the neurochemistry is remarkably complex for such a simple circuit/behaviour. This suggests that the pharyngeal activity may be subject to exquisitely precise regulation depending on the animal’s environment and status. This therefore provides a very tractable genetic model to investigate neural mechanisms for signal integration and synaptic plasticity in a well-defined neuronal network that directs a quantifiable behaviour, feeding.     

On the Mechanism of Synaptic Depression Induced by CaMKIIN,an Endogenous Inhibitor of CaMKII

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca²⁺ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.