A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.     

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.     

Disrupted-in-schizophrenia (DISC1) functions presynaptically at glutamatergic synapses

The pathophysiology of schizophrenia is believed to involve defects in synaptic transmission, and the function of many schizophrenia-associated genes, including DISC1, have been linked to synaptic function at glutamatergic synapses. Here we develop a rodent model via in utero electroporation to assay the presynaptic function of DISC1 at glutamatergic synapses. We used a combination of mosaic transgene expression, RNAi knockdown and optogenetics to restrict both genetic manipulation and synaptic stimulation of glutamatergic neurons presynaptic to other layer 2/3 neocortical pyramidal neurons that were then targeted for whole-cell patch-clamp recording. We show that expression of the DISC1 c-terminal truncation variant that is associated with Schizophrenia alters the frequency of mEPSCs and the kinetics of evoked glutamate release. In addition, we show that expression level of DISC1 is correlated with the probability of glutamate release such that increased DISC1 expression results in paired-pulse depression and RNAi knockdown of DISC1 produces paired-pulse facilitation. Overall, our results support a direct presynaptic function for the schizophrenia-associated gene, DISC1.     

Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse

We have systematically screened EMS-mutagenized Drosophila for embryonic lethal strains with defects in glutamatergic synaptic transmission. Surprisingly, this screen led to the identification of several alleles with missense mutations in highly conserved regions of Dgad1. Analysis of these gad mutants reveals that they are paralyzed owing to defects in glutamatergic transmission at the neuromuscular junction. Further electrophysiological and immunohistochemical examination reveals that these mutants have greatly reduced numbers of postsynaptic glutamate receptors in an otherwise morphologically normal synapse. By overexpressing wild-type Dgad1 in selected neurons, we show that GAD is specifically required in the presynaptic neuron to induce a postsynaptic glutamate receptor field, and that the level of postsynaptic receptors is closely dependent on presynaptic GAD function. These data demonstrate that GAD plays an unexpected role in glutamatergic synaptogenesis.     

Membrane topology of the Drosophila vesicular glutamate transporter

The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. Here we present data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, we have determined the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein. Using immunoisolation, we have identified complementary tagged sites that face the cytoplasm. Our data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins.     

Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila.

Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing beta-galactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the leucine-rich repeat gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition.     

Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.     

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.     

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.     

Targeted gene expression by the Gal4-UAS system in zebrafish

Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila . On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.     

Assessing the roles of glutamatergic and cholinergic synaptic drive in the control of fictive swimming frequency in young Xenopus tadpoles

This paper investigates the proposal that the frequency of the swimming central pattern generator in young Xenopus tadpoles is partly determined by the population of glutamatergic premotor interneurons active on each cycle. During fictive swimming spinal neurons also receive cholinergic and electrotonic excitation from motoneurons. As frequency changes during swimming we make two predictions: first, since most motoneurons fire very reliably at all frequencies, the electrotonic and nicotinic drive from motoneurons should remain constant, and second, when swimming frequency decreases, the glutamatergic drive should decrease as the number of active premotor excitatory interneurons decreases. We have tested these predictions by measuring the excitatory synaptic drive to motoneurons as frequency changes during fictive swimming. The components of synaptic drive were revealed by the local microperfusion of strychnine together with different excitatory antagonists. After blocking the nicotinic acetylcholine receptor, the mainly glutmatergic excitatory synaptic drive still changed with frequency. However, when glutamate receptors or all chemical transmission was blocked, excitation did not change with frequency. Our predictions are confirmed, suggesting that premotor excitatory interneurons are a major factor in frequency control in the tadpole central pattern generator and that motoneurons provide a stable background excitation. Accepted: 14 August 1998     

Down-regulation of vesicular glutamate transporters precedes cell loss and pathology in Alzheimer's disease

Alzheimer's disease (AD) is characterized pathologically by plaques, tangles, and cell and synapse loss. As glutamate is the principle excitatory neurotransmitter of the CNS, the glutamatergic system may play an important role in AD. An essential step in glutamate neurotransmission is the concentration of glutamate into synaptic vesicles before release from the presynaptic terminal. Recently a group of proteins responsible for uptake has been identified - the vesicular glutamate transporters (VGLUTs). The generation of antibodies has facilitated the study of glutamatergic neurones. Here, we used antibodies to the VGLUTs together with immunohistochemistry and western blotting to investigate the status of glutamatergic neurones in temporal, parietal and occipital cortices of patients with AD; these regions were chosen to represent severely, moderately and mildly affected regions at the end stage of the disease. There was no change in expression of the synaptic markers in relation to total protein in the temporal cortex, but a significant reduction in synaptophysin and VGLUT1 was found in both the parietal and occipital cortices. These changes were found to relate to the number of tangles in the temporal cortex. There were no correlations with either mental test score or behaviour syndromes, with the exception of depression.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA(B) type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA(B) receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA(A) receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in alpha-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.     

Into great silence without VGLUT3

The vesicular glutamate transporters VGLUT1 and VGLUT2 fill synaptic vesicles with glutamate, an essential prerequisite for glutamatergic transmission in the CNS. In contrast, the third isoform, VGLUT3, is not confined to glutamatergic neurons, and its function has remained enigmatic. In this issue of Neuron, Seal et al. show that mice lacking VGLUT3 are profoundly deaf and exhibit nonconvulsive seizures.     

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l-glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l-glutamate in synaptic-like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l-glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT-PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co-localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l-glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.     

Drosophila melanogaster chemosensory and muscle development: Identification and properties of a novel allele ofscalloped and of a new locus, SG18.1, in a Gal4 enhancer trap screen

Our primary interest is to probe into the genetic and molecular mechanisms underlying the development of the chemosensory and neuromuscular systems inDrosophila melanogaster. We have generated and characterized 40 Gal4 enhancer trap lines with P-Gal4 insertion as an attempt to identify genes with a likely role in the development and differentiation of chemosensory and neuromuscular tissues, and at the same time to obtain Gal4 drivers that would facilitate targeted ectopic expression of genes in these tissues. Insertion strain SG18.1 has reporter gene activity in major olfactory components of the adult fly and in their presumptive areas in the imaginal discs. SG29.1 has an insertion in thescalloped gene and has been useful in understanding genetic interactions that pattern the wing and in defining the role ofscalloped in muscle development in flies.