A tripartite synapse model in Drosophila

Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function.     

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.     

A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult

Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission.     

Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors

Homeostatic plasticity may compensate for Hebbian forms of synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), by scaling neuronal output without changing the relative strength of individual synapses. This delicate balance between neuronal output and distributed synaptic weight may be necessary for maintaining efficient encoding of information across neuronal networks. Here, we demonstrate that Arc/Arg3.1, an immediate-early gene (IEG) that is rapidly induced by neuronal activity associated with information encoding in the brain, mediates homeostatic synaptic scaling of AMPA type glutamate receptors (AMPARs) via its ability to activate a novel and selective AMPAR endocytic pathway. High levels of Arc/Arg3.1 block the homeostatic increases in AMPAR function induced by chronic neuronal inactivity. Conversely, loss of Arc/Arg3.1 results in increased AMPAR function and abolishes homeostatic scaling of AMPARs. These observations, together with evidence that Arc/Arg3.1 is required for memory consolidation, reveal the importance of Arc/Arg3.1's dynamic expression as it exerts continuous and precise control over synaptic strength and cellular excitability.     

Astrocytes in endocannabinoid signalling

Astrocytes are emerging as integral functional components of synapses, responding to synaptically released neurotransmitters and regulating synaptic transmission and plasticity. Thus, they functionally interact with neurons establishing tripartite synapses: a functional concept that refers to the existence of communication between astrocytes and neurons and its crucial role in synaptic function. Here, we discuss recent evidence showing that astrocytes are involved in the endocannabinoid (ECB) system, responding to exogenous cannabinoids as well as ECBs through activation of type 1 cannabinoid receptors, which increase intracellular calcium and stimulate the release of glutamate that modulates synaptic transmission and plasticity. We also discuss the consequences of ECB signalling in tripartite synapses on the astrocyte-mediated regulation of synaptic function, which reveal novel properties of synaptic regulation by ECBs, such as the spatially controlled dual effect on synaptic strength and the lateral potentiation of synaptic efficacy. Finally, we discuss the potential implications of ECB signalling for astrocytes in brain pathology and animal behaviour.     

Glutamate and GABA receptors in vertebrate glial cells

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.     

Dissecting tripartite synapses with STED microscopy

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.     

神经胶质细胞与突触可塑性研究新进展

突触的可塑性是研究学习与记忆的基础,很长时间以来人们对突触的可塑性研究主要集中在神经元和突触上;而胶质细胞的作用较少受到注意。最近的研究发现胶质细胞也参与突触的构成并影响突触的活动。研究表明中枢神经系统中的胶质细胞包括星形胶质细胞、小胶质细胞和少突胶质细胞可分别通过谷氨酸、丝氨酸、甘氨酸、ATP等信号调节突触的可塑性,从而为突触的可塑性研究提供了新的思路和方向,并有助于阐明突触的发生以及学习与记忆的机制。     

Remodeling of astrocytes, a prerequisite for synapse turnover in the adult brain? Insights from the oxytocin system of the hypothalamus

Neurons, including their synapses, are generally ensheathed by fine processes of astrocytes, but this glial coverage can be altered under different physiological conditions that modify neuronal activity. Changes in synaptic connectivity accompany astrocytic transformations so that an increased number of synapses are associated with reduced astrocytic coverage of postsynaptic elements, whereas synaptic numbers are reduced on reestablishment of glial coverage. A system that exemplifies activity-dependent structural synaptic plasticity in the adult brain is the hypothalamo-neurohypophysial system, and in particular, its oxytocin component. Under strong, prolonged activation (parturition, lactation, chronic dehydration), extensive portions of somatic and dendritic surfaces of magnocellular oxytocin neurons are freed of intervening astrocytic processes and become directly juxtaposed. Concurrently, they are contacted by an increased number of inhibitory and excitatory synapses. Once stimulation is over, astrocytic processes again cover oxytocinergic surfaces and synaptic numbers return to baseline levels. Such observations indicate that glial ensheathment of neurons is of consequence to neuronal function, not only directly, for example by modifying synaptic transmission, but indirectly as well, by preparing neuronal surfaces for synapse turnover.     

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

The Influence of Synaptic Weight Distribution on Neuronal Population Dynamics

The manner in which different distributions of synaptic weights onto cortical neurons shape their spiking activity remains open. To characterize a homogeneous neuronal population, we use the master equation for generalized leaky integrate-and-fire neurons with shot-noise synapses. We develop fast semi-analytic numerical methods to solve this equation for either current or conductance synapses, with and without synaptic depression. We show that its solutions match simulations of equivalent neuronal networks better than those of the Fokker-Planck equation and we compute bounds on the network response to non-instantaneous synapses. We apply these methods to study different synaptic weight distributions in feed-forward networks. We characterize the synaptic amplitude distributions using a set of measures, called tail weight numbers, designed to quantify the preponderance of very strong synapses. Even if synaptic amplitude distributions are equated for both the total current and average synaptic weight, distributions with sparse but strong synapses produce higher responses for small inputs, leading to a larger operating range. Furthermore, despite their small number, such synapses enable the network to respond faster and with more stability in the face of external fluctuations.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Substantial changes in synaptic firing frequencies induced by glial ATP hysteresis

Recent experimental studies strongly suggest the influence of glial purinergic transmission in the modulation of synaptic dynamics. By releasing adenosine triphosphate (ATP), which accumulates as adenosine, astrocytes tonically suppressed synaptic transmission. The delayed multi-step feedback of the glial ATP with the neuron suggests the existence of a discrete hysteresis phenomena. By integration of this hysteretic behavior into a delayed leaky integrate-and-fire model for the tripartite synapses, a significant sensitivity of the pre- and postsynaptic firing frequency patterns to the adenosine feedback-delays is observed that might be of importance for adenosine-related neurological deficits, such as sleep disorders.     

The specificity of platelet glutamate receptor supersensitivity in psychotic disorders

Hypoglutamatergic function is implicated in the pathogenesis of schizophrenia, and supersensitivity of platelet NMDA receptors has been reported in schizophrenia. The aim of this study was to examine the platelet glutamate receptor sensitivity in patients with schizophrenia (n=12), mania with psychotic features (n=10) and depression with psychotic features (n=10) and matched controls (n=12) in order to assess if this is a marker of schizophrenia or occurs in other psychotic conditions. Glutamate receptor sensitivity was assessed using the intracellular calcium response to glutamate measured with spectrofluorometry. The percentage response of the schizophrenic and depressed psychotic subjects to glutamate stimulation was significantly greater than control subjects (p<0.005). The mania with psychotic features group was not significantly different to controls. This data suggests that platelet glutamate receptors may be supersensitive in schizophrenia and depression with psychotic features. Furthermore, the platelet may be a possible peripheral marker of glutamate function in schizophrenia and depression with psychotic features.     

Astrocyte-dependent circuit remodeling by synapse phagocytosis

In the central nervous system, synaptic pruning, the removal of unnecessary synaptic contacts, is an essential process for proper circuit maturation in neurodevelopment as well as for synaptic homeostasis in the adult stage. Dysregulation of synaptic pruning can contribute to the initiation and progression of various mental disorders, such as schizophrenia and depression, as well as neurodegenerative diseases including Alzheimer's disease. In the past 15 years, pioneering works have demonstrated that different types of glial cells regulate the number of synapses by selectively eliminating them through phagocytic molecular machinery. Although a majority of findings have been focused on microglia, it is increasingly evident that astrocytes function as a critical player in activity-dependent synapse elimination in developing, adult, and diseased brains. In this review, we will discuss recent findings showing the mechanisms and physiological importance of astrocyte-mediated synapse elimination in controlling synapses and circuit homeostasis. We propose that astrocytes play dominant and non-redundant roles in eliminating synapses during the activity-dependent circuit remodeling processes that do not involve neuro-inflammation.     

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non‐synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high‐resolution biochemical analyses of specific synapse subpopulations. Employing knock‐in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.     

Glial cells

The nervous system is built from two broad categories of cells, neurones and glial cells. The glial cells outnumber the neurones and the two cell types occupy a comparable amount of space in nervous tissue. The main glial cell types are, in the central nervous system, astrocytes and oligodendrocytes and, in the peripheral nervous system, Schwann cells, enteric glial cells and satellite cells. In the embryo, glial cells form a cellular framework that permits the development of the rest of the nervous system, and regulate neuronal survival and differentiation. The best known function of glia in the adult is the formation of myelin sheaths around axons thus allowing the fast conduction of signalling essential for nervous system function. Glia also maintain appropriate concentrations of ions and neurotransmitters in the neuronal environment. Increasing body of evidence indicates that glial cells are essential regulators of the formation, maintenance and function of synapses, the key functional unit of the nervous system.     

Acquiring new information in a neuronal network: from Hebb's concept to homeostatic plasticity

Synaptic plasticity is the cellular mechanism underlying the phenomena of learning and memory. Much of the research on synaptic plasticity is based on the postulate of Hebb (1949) who proposed that, when a neuron repeatedly takes part in the activation of another neuron, the efficacy of the connections between these neurons is increased. Plasticity has been extensively studied, and often demonstrated through the processes of LTP (Long Term Potentiation) and LTD (Long Term Depression), which represent an increase and a decrease of the efficacy of long-term synaptic transmission. This review summarizes current knowledge concerning the cellular mechanisms of LTP and LTD, whether at the level of excitatory synapses, which have been the most studied, or at the level of inhibitory synapses. However, if we consider neuronal networks rather than the individual synapses, the consequences of synaptic plasticity need to be considered on a large scale to determine if the activity of networks are changed or not. Homeostatic plasticity takes into account the mechanisms which control the efficacy of synaptic transmission for all the synaptic inputs of a neuron. Consequently, this new concept deals with the coordinated activity of excitatory and inhibitory networks afferent to a neuron which maintain a controlled level of excitability during the acquisition of new information related to the potentiation or to the depression of synaptic efficacy. We propose that the protocols of stimulation used to induce plasticity at the synaptic level set up a "homeostatic potentiation" or a "homeostatic depression" of excitation and inhibition at the level of the neuronal networks. The coordination between excitatory and inhibitory circuits allows the neuronal networks to preserve a level of stable activity, thus avoiding episodes of hyper- or hypo-activity during the learning and memory phases.     

Dressed neurons: modeling neural-glial interactions

Based on recent experimental data, we design a model for neuronal membrane potentials that incorporates the influence of the surrounding glia (dressed neurons). A neurotransmitter released into the synaptic cleft triggers a Ca(2+) response in nearby glial cells that spreads as a Ca(2+) wave and interacts with other synapses via the release of glutamate from astrocytes. We consider the simple case of a neuron-glia circuit that consists of a single neuron that triggers a Ca(2+) response in the glial cell which in turn feeds back into synapses of the same neuron. It is shown that persistent spiking can occur if the glutamate receptors on the astrocytes are overexpressed--a condition that has been reported from patients suffering from mesial-lobe epilepsy.     

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.