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.     

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.     

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.     

The expression of vesicular glutamate transporters defines two classes of excitatory synapse

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

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.     

Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine

Dopamine neurons have been suggested to use glutamate as a cotransmitter. To identify the basis of such a phenotype, we have examined the expression of the three recently identified vesicular glutamate transporters (VGLUT1-3) in postnatal rat dopamine neurons in culture. We found that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3. In comparison, serotonin neurons express only VGLUT3. Single-cell RT-PCR experiments confirmed the presence of VGLUT2 mRNA in dopamine neurons. Arguing for phenotypic heterogeneity among axon terminals, we find that only a proportion of terminals established by dopamine neurons are VGLUT2-positive. Taken together, our results provide a basis for the ability of dopamine neurons to release glutamate as a cotransmitter. A detailed analysis of the conditions under which DA neurons gain or loose a glutamatergic phenotype may provide novel insight into pathophysiological processes that underlie diseases such as schizophrenia, Parkinson's disease and drug dependence.     

Role of glutamate in locomotor rhythm generating neuronal circuitry

Central pattern generators (CPGs) are defined as neuronal circuits capable of producing a rhythmic and coordinated output without the influence of sensory input. The locomotor and respiratory neuronal circuits are two of the better-characterized CPGs, although much work remains to fully understand how these networks operate. Glutamatergic neurons are involved in most neuronal circuits of the nervous system and considerable efforts have been made to study glutamate receptors in nervous system signaling using a variety of approaches. Because of the complexity of glutamate-mediated signaling and the variety of receptors triggered by glutamate, it has been difficult to pinpoint the role of glutamatergic neurons in neuronal circuits. In addition, glutamate is an amino acid used by every cell, which has hampered identification of glutamatergic neurons. Glutamatergic excitatory neurotransmission is dependent on the release from glutamate-filled presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Recent data describe that Vglut2 (Slc17a6) null mutant mice die immediately after birth due to a complete loss of the stable autonomous respiratory rhythm generated by the pre-B?tzinger complex. Surprisingly, we found that basal rhythmic locomotor activity is not affected in Vglut2 null mutant embryos. With this perspective, we discuss data regarding presence of VGLUT1, VGLUT2 and VGLUT3 positive neuronal populations in the spinal cord.     

Expression and localization of vesicular glutamate transporters in pancreatic islets, upper gastrointestinal tract, and testis.

The wide-ranging expression of glutamate receptors in peripheral tissues suggests an unexpectedly wider role(s) of l-glutamate as an intercellular signaling molecule. However, the peripheral glutamatergic system is poorly understood, partly because the sites of l-glutamate signal appearance are less well characterized. Vesicular glutamate transporters (VGLUTs) are potential probes for the sites of vesicular storage and subsequent secretion of l-glutamate. In this study we raised specific polyclonal antibodies against two VGLUT isoforms, VGLUT1 and VGLUT2, and investigated their localization in peripheral tissues of rat. We detected the expression of either VGLUT1 or VGLUT2, or both, in pancreas, stomach, intestine, and testis. In pancreas, VGLUT1 and VGLUT2 are present in pancreatic polypeptide-containing secretory granules in F-cells in the islets of Langerhans. In stomach, VGLUT2 is abundant in the antrum and pylorus and is present in a subset of pancreatic polypeptide-containing cells. In intestine, VGLUT2 is abundant in the ileum and is co-localized with glucagon-like immunoreactive peptide and polypeptide YY (PYY). In testis, VGLUT2 is expressed and localized in the outer acrosomal membrane of spermatids, where KA1 and GluR5, kainate receptor subunits, are almost always localized. Taken together, these results strongly suggest the occurrence of a peripheral glutamatergic system in the gastroenteropancreatic system and testis.     

Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia

The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.     

Up-regulation in expression of vesicular glutamate transporter 3 in substantia nigra but not in striatum of 6-hydroxydopamine-lesioned rats

Overactivity of the glutamatergic system is suggested to be closely related to the onset and pathogenesis of Parkinson's disease. Vesicular glutamate transporters (VGLUT1, T2 and T3) are a group of glutamate transporters in neurons that are responsible for transporting glutamate into synaptic vesicles and they are key elements for homeostasis of glutamate neurotransmission. The present study was aimed to investigate the expression of VGLUT1, T2 and T3 proteins after the onset of Parkinson's disease. A rat model of Parkinson's disease, the 6-hydroxydopamine-lesioned rat, was employed. Immunocytochemistry revealed that VGLUT1, T2 and T3 immunoreactivity was not modulated in the striatum of the lesioned rat. Western blotting analyses also showed that there was no change in the expression of T1, T2 and T3 proteins in the striatum. In contrast, no VGLUT1 protein was detected in the substantia nigra. After the lesion, levels of VGLUT2 immunoreactivity and protein were not modulated. Significant increase of VGLUT3 immunoreactivity was observed in the perikarya of GABAergic substantia nigra pars reticulata neurons (+14.7%) although VGLUT3 protein was not modulated in the nigral tissues. VGLUT3 in GABAergic neurons is suggested to play a role in GABA synthesis. The present results may therefore implicate that VGLUT1 and T2 are not modulated in the striatum and the substantia nigra of the 6-hydroxydopamine-lesioned rat and only VGLUT3 plays a role in pathogenesis of Parkinson's disease.     

Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3

The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.     

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。     

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.     

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.     

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.     

Tetrapeptide inhibitors of the glutamate vesicular transporter (VGLUT)

Quinoline-2,4-dicarboxylic acids (QDCs) bearing lipophilic substituents in the 6- or 7-position were shown to be inhibitors of the glutamate vesicular transporter (VGLUT). Using the arrangement of the QDC lipophilic substituents as a template, libraries of X(1)X(2)EF and X(1)X(2)EW tetrapeptides were synthesized and tested as VGLUT inhibitors. The peptides QIEW and WNEF were found to be the most potent. Further stereochemical deconvolution of these two peptides showed dQlIdElW to be the best inhibitor (K(i)=828+/-252 microM). Modeling and overlay of the tetrapeptide inhibitors with the existing pharmacophore showed that H-bonding and lipophilic residues are important for VGLUT binding.     

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.