VGLUTs: 'exciting' times for glutamatergic research?

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system (CNS). Glutamate is first synthesized in the cytoplasm of presynaptic terminals before being loaded into synaptic vesicles, which fuse with the plasma membrane, releasing their contents, in response to neuronal activity. The important process of synaptic vesicle loading is mediated by a transport protein, collectively known as vesicular glutamate transporter (VGLUT). Controlling the activity of these transporters could potentially modulate the efficacy of glutamatergic neurotransmission. In recent years, three isoforms of mammalian VGLUTs have been cloned and molecularly characterized in detail. Probing these three VGLUTs has been proven to be the most reliable way of visualizing sites of glutamate release in the mammalian CNS. Immunohistochemical studies on VGLUTs suggest that glutamatergic neurons are categorized into subgroups depending on which VGLUT isoform they contain. Recent studies on VGLUT1-deficient mice have led various models to be postulated concerning the possible roles of VGLUTs in synaptic physiology, such as presynaptic regulation of quantal size and activity-dependent short-term plasticity.     

The inorganic phosphate transport system in the brain and the potency as vesicular glutamate transporters

In human and rat brains, two highly homologous proteins have been identified as brain inorganic phosphate cotransport systems: a brain-specific sodium-dependent inorganic phosphate cotransporter (BNPI) and a differentiation-associated sodium-dependent inorganic phosphate cotransporter (DNPI). However, the current evidence shows that the primary role of BNPI is to accumulate excitatory amino acid neurotransmitter glutamate into synaptic vesicles as a vesicular transporter. This is indicated by an experiment in which rBNPI cDNA-transfected PC12 cells show a highly selective uptake of L-glutamate. Taken the 82% amino acid identity of DNPI to BNPI into consideration, it is very likely that DNPI is another kind of vesicular transporter for glutamate and/or related amino acids. An earlier study on BNPI expression in rat and human forebrains describes that its gene message is almost exclusively demonstrated in the telencephalon, particularly in glutamatergic projection neurons of the cerebral cortex and hippocampus. In contrast, the regional distribution of DNPI gene message is quite different from that of BNPI in the human brain, where the DNPI gene message is largely restricted to the diencephalon, especially in the thalamus. Many neurons in the dorsal thalamus are known as glutamatergic projection neurons giving rise to the thalamocortical and/or thalamostriatal pathways. Thus, both transporter proteins would be much better neuroanatomical markers than previously utilized to identify glutamatergic neurons and to distinguish distinct subpopulations of the identified neurons. Thus, the old understanding on the organization and function of glutamatergic neuron system should be re-evaluated on the basis of the two vesicular glutamate transporter proteins. Along with this thinking, we have recently analyzed the cellular and regional localization of DNPI in the rat brain by in situ hybridization and immunohistochemistry. In this review article, we explained our results about the regional difference between DNPI and BNPI in the rat forebrain, and discussed the possible functional significance of DNPI.     

Vesicular glutamate transporters (VGLUTs): the three musketeers of glutamatergic system

Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) and glutamatergic transmission is critical for controlling neuronal activity. Glutamate is stored in synaptic vesicles and released upon stimulation. The homeostasis of glutamatergic system is maintained by a set of transporters present in plasma membrane and in the membrane of synaptic vesicles. The family of vesicular glutamate transporters in mammals is comprised of three highly homologous proteins: VGLUT1-3. The expression of particular VGLUTs is largely complementary with limited overlap and so far they are most specific markers for neurons that use glutamate as neurotransmitter. VGLUTs are regulated developmentally and determine functionally distinct populations of glutamatergic neurons. Controlling the activity of these proteins could potentially modulate the efficiency of excitatory neurotransmission. This review summarizes the recent knowledge concerning molecular and functional characteristic of vesicular glutamate transporters, their development, contribution to synaptic plasticity and their involvement in pathology of the nervous system.     

Vesicular glutamate transporter 1 immunostaining in the normal and epileptic human cerebral cortex

Glutamate is the main excitatory neurotransmitter in the brain where, due to the activity of specific vesicular glutamate transporters, it accumulates in synaptic vesicles. The vesicular glutamate transporter 1 is found in the majority of axon terminals that form asymmetrical (excitatory) synapses in the rat neocortex. However, since there is no information available regarding the distribution of vesicular glutamate transporter 1 in the human neocortex, we have used correlative light and electron microscopy to define its expression in this tissue. We found that the distribution of vesicular glutamate transporter 1-immunoreactivity is virtually identical to that found in the rat neocortex, both at the light and electron microscope levels. Therefore, we assessed whether vesicular glutamate transporter 1 immunostaining might be a useful tool to study the pathological alterations of glutamatergic transmission in the epileptic cerebral cortex. We analyzed the distribution of vesicular glutamate transporter 1 in the peritumoral neocortex of patients with epilepsy secondary to low-grade tumors. In these regions, we found alterations in the pattern of vesicular glutamate transporter 1-immunoreactivity that perfectly matched the neuronal loss and gliosis, as well as the decrease in the number of asymmetrical synapses identified by electron microscopy in this tissue. Thus, vesicular glutamate transporter 1 immunostaining appears to be a reliable and simple tool to study glutamatergic synapses in the normal and epileptic human cerebral cortex.     

The SLC32 transporter,a key protein for the synaptic release of inhibitory amino acids

The SLC32 family comprises a single member: the vesicular inhibitory amino acid transporter (VIAAT) or vesicular GABA transporter (VGAT). It belongs to a eukaryotic-specific superfamily of H(+)-coupled amino acid transporters, which also comprises the mammalian SLC36 and SLC38 transporters. VIAAT exchanges GABA or glycine for protons. It is present on synaptic vesicles of GABAergic and glycinergic neurons, and in some endocrine cells, where it ensures the H(+)-ATPase-driven uptake, and subsequent exocytotic release, of inhibitory amino acids. Despite a similar function in vesicular neurotransmitter loading, VIAAT is not related to the vesicular glutamate transporter (VGLUT, SLC17) or the vesicular monoamine transporter/vesicular acetylcholine transporter (VMAT/VACHT, SLC18) proteins.     

Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract

The nucleus of the solitary tract is the site of termination of primary afferent fibers running in the facial, glossopharyngeal and vagus nerves. The present study was performed to map the distribution of glutamatergic axons terminals in the rat nucleus of the solitary tract using immunodetection of vesicular glutamate transporter 1 and vesicular glutamate transporter 2. The two vesicular glutamate transporters were differentially distributed among nucleus of the solitary tract subdivisions. Vesicular glutamate transporter 1 immunoreactivity was mostly found in the lateral part of the nucleus (ventrolateral, interstitial and intermediate subdivisions) whereas vesicular glutamate transporter 2 labeling was distributed throughout the nucleus of the solitary tract. Electron microscope examination indicated that vesicular glutamate transporter immunoreactivity was localized in axon terminals filled with round synaptic vesicles. After injection of cholera toxin B subunit in sensory ganglia, anterograde labeling was found in vesicular glutamate transporter 1, as well as vesicular glutamate transporter 2-immunoreactive boutons. Double immunolabeling experiments allowed distinctions between terminals expressing either vesicular glutamate transporter 1 or vesicular glutamate transporter 2 or both vesicular glutamate transporter 1 and vesicular glutamate transporter 2 immunoreactivities. The latter population, expressing both transporters immunolabeling, completely disappeared after deafferentation induced by removal of sensory ganglia. This study indicates that vesicular glutamate transporter content identifies three different subpopulations of glutamatergic boutons in the nucleus of the solitary tract and provides definitive evidence that primary afferent neurons contribute glutamatergic terminals to the nucleus of the solitary tract.     

Synaptic vesicle protein trafficking at the glutamate synapse

Expression of the integral and associated proteins of synaptic vesicles is subject to regulation over time, by region, and in response to activity. The process by which changes in protein levels and isoforms result in different properties of neurotransmitter release involves protein trafficking to the synaptic vesicle. How newly synthesized proteins are incorporated into synaptic vesicles at the presynaptic bouton is poorly understood. During synaptogenesis, synaptic vesicle proteins sort through the secretory pathway and are transported down the axon in precursor vesicles that undergo maturation to form synaptic vesicles. Changes in protein content of synaptic vesicles could involve the formation of new vesicles that either mix with the previous complement of vesicles or replace them, presumably by their degradation or inactivation. Alternatively, new proteins could individually incorporate into existing synaptic vesicles, changing their functional properties. Glutamatergic vesicles likely express many of the same integral membrane proteins and share certain common mechanisms of biogenesis, recycling, and degradation with other synaptic vesicles. However, glutamatergic vesicles are defined by their ability to package glutamate for release, a property conferred by the expression of a vesicular glutamate transporter (VGLUT). VGLUTs are subject to regional, developmental, and activity-dependent changes in expression. In addition, VGLUT isoforms differ in their trafficking, which may target them to different pathways during biogenesis or after recycling, which may in turn sort them to different vesicle pools. Emerging data indicate that differences in the association of VGLUTs and other synaptic vesicle proteins with endocytic adaptors may influence their trafficking. These observations indicate that independent regulation of synaptic vesicle protein trafficking has the potential to influence synaptic vesicle protein composition, the maintenance of synaptic vesicle pools, and the release of glutamate in response to changing physiological requirements.     

Immunohistochemical evidence for synaptic release of glutamate from orexin terminals in the locus coeruleus

Orexin (Orx or hypocretin) is critically important for maintaining wakefulness, since in its absence, narcolepsy with cataplexy occurs. In this role, Orx-containing neurons can exert their influence upon multiple targets through the brain by release of Orx but possibly also by release of other neurotransmitters. Indeed, evidence was previously presented to suggest that Orx terminals could utilize glutamate (Glu) in addition to Orx as a neurotransmitter. Using fluorescence and confocal laser scanning microscopy, we investigated whether Orx varicosities contain the presynaptic markers for synaptic release of Glu or GABA and come into contact with postsynaptic markers for excitatory synapses within the locus coeruleus of the rat brain. We found that a proportion of the Orx+ varicosities were immunostained for the vesicular transporter for Glu, VGluT2. None were immunostained for vesicular glutamate transporter 1 (VGluT1) or VGluT3 or for the vesicular transporter for GABA, vesicular GABA transporter (VGAT). Among the Orx+ varicosities, 4% of all and 28% of large varicosities contained VGluT2. A similar proportion of the large Orx+ varicosities contained synaptophysin (Syp), a presynaptic marker for synaptic vesicles. Orx+ varicosities also contacted elements immunostained for postsynaptic density protein-95 (PSD)-95, a postsynaptic marker for glutamatergic synapses. We thus conclude that synaptic release of Glu occurs from Orx terminals within the locus coeruleus and can thus be important for the engagement of noradrenergic neurons in stimulating and maintaining arousal.     

Functional expression of two system A glutamine transporter isoforms in rat auditory brainstem neurons

Glutamine plays multiple roles in the CNS, including metabolic functions and production of the neurotransmitters glutamate and GABA. It has been proposed to be taken up into neurons via a variety of membrane transport systems, including system A, which is a sodium-dependent electrogenic amino acid transporter system. In this study, we investigate glutamine transport by application of amino acids to individual principal neurons of the medial nucleus of the trapezoid body (MNTB) in acutely isolated rat brain slices. A glutamine transport current was studied in patch-clamped neurons, which had the electrical and pharmacological properties of system A: it was sodium-dependent, had a non-reversing current-voltage relationship, was activated by proline, occluded by N-(methylamino)isobutyric acid (MeAIB), and was unaffected by 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH). Additionally, we examined the expression of different system A transporter isoforms using immunocytochemical staining with antibodies raised against system A transporter 1 and 2 (SAT1 and SAT2). Our results indicate that both isoforms are expressed in MNTB principal neurons, and demonstrate that functional system A transporters are present in the plasma membrane of neurons. Since system A transport is highly regulated by a number of cellular signaling mechanisms and glutamine then goes on to activate other pathways, the study of these transporters in situ gives an indication of the mechanisms of neuronal glutamine supply as well as points of regulation of neurotransmitter production, cellular signaling and metabolism in the native neuronal environment.     

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals.From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.     

Differential expression of vesicular glutamate transporters 1 and 2 may identify distinct modes of glutamatergic transmission in the macaque visual system

Glutamate is the primary neurotransmitter utilized by the mammalian visual system for excitatory neurotransmission. The sequestration of glutamate into synaptic vesicles, and the subsequent transport of filled vesicles to the presynaptic terminal membrane, is regulated by a family of proteins known as vesicular glutamate transporters (VGLUTs). Two VGLUT proteins, VGLUT1 and VGLUT2, characterize distinct sets of glutamatergic projections between visual structures in rodents and prosimian primates, yet little is known about their distributions in the visual system of anthropoid primates. We have examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the visual system of macaque monkeys, an Old World anthropoid primate, in order to determine their relative distributions in the superior colliculus, lateral geniculate nucleus, pulvinar complex, V1 and V2. Distinct expression patterns for both VGLUT1 and VGLUT2 identified architectonic boundaries in all structures, as well as anatomical subdivisions of the superior colliculus, pulvinar complex, and V1. These results suggest that VGLUT1 and VGLUT2 clearly identify regions of glutamatergic input in visual structures, and may identify common architectonic features of visual areas and nuclei across the primate radiation. Additionally, we find that VGLUT1 and VGLUT2 characterize distinct subsets of glutamatergic projections in the macaque visual system; VGLUT2 predominates in driving or feedforward projections from lower order to higher order visual structures while VGLUT1 predominates in modulatory or feedback projections from higher order to lower order visual structures. The distribution of these two proteins suggests that VGLUT1 and VGLUT2 may identify class 1 and class 2 type glutamatergic projections within the primate visual system (Sherman and Guillery, 2006).     

Presynaptic regulation of quantal size: K+/H+ exchange stimulates vesicular glutamate transport

The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δμ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δμ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δμ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.     

Stable expression of the vesicular GABA transporter following photothrombotic infarct in rat brain

Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.     

GABA(B) receptors at glutamatergic synapses in the rat striatum

Although multiple effects of GABA(B) receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABA(B) receptor subunits, GABA(B1) and GABA(B2). In addition, to investigate the relationship between GABA(B) receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABA(B1) and GABA(B2) was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABA(B) receptor subunits and vesicular glutamate transporters revealed that labeling for both GABA(B1) and GABA(B2) was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABA(B) heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABA(B) autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABA(B) receptors.     

A genetic interaction between the vesicular acetylcholine transporter VAChT/UNC-17 and synaptobrevin/SNB-1 in C. elegans

Acetylcholine, a major excitatory neurotransmitter in Caenorhabditis elegans, is transported into synaptic vesicles by the vesicular acetylcholine transporter encoded by unc-17. The abnormal behavior of unc-17(e245) mutants, which have a glycine-to-arginine substitution in a transmembrane domain, is markedly improved by a mutant synaptobrevin with an isoleucine-to-aspartate substitution in its transmembrane domain. These results suggest an association of vesicular soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) components with vesicular neurotransmitter transporters.     

Immunocytochemical evidence of vesicular localization of the orphan transporter RXT1 in the rat spinal cord.

Rxt1, a member of the Na+/Cl- orphan transporter family, exhibits numerous features suggesting a role as plasma membrane transporter. Despite numerous attempts, its substrate has not yet been identified, although immunocytochemical studies have shown that Rxt1 distribution generally matches that of glutamate or GABA. In order to further characterize Rxt1, its detailed immunocytochemical distribution in the rat spinal cord and dorsal root ganglia was studied at both light microscope and ultrastructural levels. The widespread distribution of Rxt1 in spinal cord and ganglia cannot be correlated with any known classical or peptidergic transmitter. Rxt1 is expressed in a subpopulation of glutamatergic primary afferent fibers, in large and medium-sized ganglion cells, while small glutamate cells exhibit generally no Rxt1-like immunoreactivity. In the spinal cord, Rxt1-immunoreactive cell body distribution is quite ubiquitous since Rxt1 is expressed in all laminae in various neuronal types like interneurons, some projection neurons and motoneurons. Some of these neurons are cholinergic. At the electron microscope level, the peroxidase labeling was never localized to the plasma membrane, but rather associated with different organelles including the outer membrane of small synaptic vesicles and large granular vesicles. This localization resembles that of vesicular transporters detected with the same method and suggests that Rxt1, in contrast to other Na+/Cl- transporters, is expressed on vesicles. This was confirmed using a pre-embedding silver-intensified colloidal gold method. Indeed, most gold particles appeared to be localized into the axoplasm on synaptic vesicle accumulations; only few gold particles were observed close to the plasma membrane. These results suggest that Rxt1, despite its molecular characteristics predicting a plasma membrane localization, might be a vesicular transporter.     

Glutamate in cancers: from metabolism to signaling

Glutamine and glutamate are major bioenergy substrates for normal and cancer cell growth. Cancer cells need more biofuel than normal tissues for energy supply, anti-oxidation activity and biomass production. Genes related to metabolic chains in many cancers are somehow mutated, which makes cancer cells more glutamate dependent. Meanwhile, glutamate is an excitatory neurotransmitter for conducting signals through binding with different types of receptors in central neuron system. Interestingly, increasing evidences have shown involvement of glutamate signaling, guided through their receptors, in human malignancy. Dysregulation of glutamate transporters, such as excitatory amino acid transporter and cystine/glutamate antiporter system, also generates excessive extracellular glutamate, which in turn, activates glutamate receptors on cancer cells and results in malignant growth. These features make glutamate an attractive target for anti-cancer drug development with some glutamate targeted but blood brain barrier impermeable anti-psychosis drugs under consideration. We discussed the relevant progressions and drawbacks in this field herein.     

Complementary distribution of vesicular glutamate transporters in the central nervous system

Two vesicular glutamate transporters (VGluTs) have been identified at the molecular level very recently and revealed to possess similar pharmacological characteristics for glutamate uptake. Vesicular glutamate transporter 1 (VGluT1), which was originally named brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), is mainly expressed in telencephalic regions, whereas vesicular glutamate transporter 2 (VGluT2), formerly referred to as differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), is produced principally in diencephalic and lower brainstem regions. Since no other proteins show as high molecular similarity to VGluT1 or VGluT2 as the two transporters exhibit, it is likely that the mammalian central nervous system use only two gene products for vesicular glutamate uptake. Immunoelectron-microscopic analysis has revealed that the two VGluTs are located on synaptic vesicles in axon terminals making an asymmetric type of synapses, supporting that they serve as vesicular transporters in excitatory terminals. Furthermore, mRNA and immunoreactivity for VGluTs are distributed largely in a complementary fashion to distinct populations of excitatory neurons; for example, in the cerebral cortex, thalamocortical axon terminals use VGluT2, whereas excitatory axon terminals of corticocortical or intracortical fibers seem to apply VGluT1 for glutamate uptake. This complementary distribution might suggest that the two VGluTs have an as yet unknown difference in functions.