Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity

Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.     

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.     

Human vesicular glutamate transporters functionally complement EAT-4 in C. elegans

The vesicular glutamate transporter (VGLUT) transports glutamate into pre-synaptic vesicles. Three isoforms of VGLUT have been identified in humans, but their functional differences remain largely unknown. EAT-4 is the only homologue of human VGLUT in C. elegans. Here we report that mutants of eat-4 exhibit hyperforaging behavior and that each of the isoforms of human VGLUT functionally rescues the defects in eat-4 worms.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

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。     

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.     

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.     

Effects of subthalamic nucleus lesions and stimulation upon corticostriatal afferents in the 6-hydroxydopamine-lesioned rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.     

Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

Background

There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis.

Results

The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group.

Conclusions

These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.     

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.     

Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment

The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1-3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.     

A role for glutamate transporters in the regulation of insulin secretion

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.     

Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100–150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9–11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles. Published: June 7, 2004.     

Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1,P 5-di(adenosine-5') pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 80 micro m) and P1,P 5-di(inosine-5') pentaphosphate (Ip5I, 100 nm), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC50 values being 21 micro m and 38 micro m for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

From glutamate co-release to vesicular synergy: vesicular glutamate transporters

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.     

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.     

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.     

Developmentally-regulated expression of tissue-specific splice variant of rat vesicular glutamate transporter 1 in retina and pineal gland

Three distinct subtypes of vesicular glutamate transporters (VGLUTs) have been identified to date that are expressed basically in a cell type-specific manner. We have found a splice variant of VGLUT1 mRNA that is expressed almost exclusively in photosensitive tissues, i.e. the retina and the pineal gland. The variant mRNA, termed VGLUT1v, contains an additional 75 base pair sequence derived from part of a second intron (designated as exon IIa) between exons 2 and 3. The variant accounted for approximately 70% and 25%of VGLUT1 mRNA in the adult retina and pineal gland, respectively. The expression of VGLUT1v was developmentally regulated in both tissues. Organ culture showed that expression of the variant in the retina increased in association with the development of rod cells, suggesting that VGLUT1v is expressed in rod cells. In situ hybridization with variant-specific probes showed expression of VGLUT1v in the inner segment layer of photoreceptor cells. On the other hand, variant expression did not parallel the development of rhodopsin-positive cells in the pineal gland. As rod cells and pinealocytes are known to release glutamate continuously at ribbon synapses, it is possible that the variant has some functional advantage over the wild-type transporter in such a specialized manner of glutamate release.