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.     

Rose Bengal analogs and vesicular glutamate transporters (VGLUTs)

Vesicular glutamate transporters (VGLUTs) allow the loading of presynaptic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. Rose Bengal (RB) is the most potent known VGLUT inhibitor (K_i 25 nM); therefore we designed, synthesized and tested in brain preparations, a series of analogs based on this scaffold. We showed that among the two tautomers of RB, the carboxylic and not the lactonic form is active against VGLUTs and generated a pharmacophore model to determine the minimal structure requirements. We also tested RB specificity in other neurotransmitter uptake systems. RB proved to potently inhibit VMAT (K_i 64 nM) but weakly VACHT (K_i >9.7 μM) and may be a useful tool in glutamate/acetylcholine co-transmission studies.     

囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布

目的观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布。方法首先采用免疫荧光三重标记I、Ⅱ型囊泡膜谷氨酸转运体和神经元核蛋白以观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布;接着注射四甲基罗达明人下颌舌骨肌神经逆行标记三叉神经运动核开口神经元,再采用免疫荧光双重标记I型囊泡膜谷氨酸转运体和神经元核蛋白以观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核开口神经元区和闭口神经元区内的分布差异。结果I型囊泡膜谷氨酸转运体阳性纤维仅在三叉神经运动核背外侧部分布,而Ⅱ型囊泡膜谷氨酸转运体阳性纤维在整个三叉神经运动核内分布;开口神经元区未观察到I型囊泡膜谷氨酸转运体阳性终末。结论闭口神经元接受I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维支配,开口神经元仅仅接受Ⅱ型囊泡膜谷氨酸转运体阳性纤维支配。     

Decreased expression of vesicular GABA transporter, but not vesicular glutamate, acetylcholine and monoamine transporters in rat brain following focal ischemia

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.     

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.     

High-density presynaptic transporters are required for glutamate removal from the first visual synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.     

Developmental pattern of three vesicular glutamate transporters in the rat superior olivary complex

Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have been identified so far, which are differentially expressed in the brain. Here, we have investigated the spatiotemporal distribution of the three VGLUTs in the rat superior olivary complex (SOC), a prominent processing center, which receives strong glutamatergic inputs and which lies within the auditory brainstem. Immunoreactivity (ir) against all three VGLUTs was found in the SOC nuclei throughout development (postnatal days P0–P60). It was predominantly seen in axon terminals, although cytoplasmic labeling also occurred. Each transporter displayed a characteristic expression pattern. In the adult SOC, VGLUT1 labeling varied from strong in the medial nucleus of the trapezoid body, lateral superior olive, and medial superior olive (MSO) to moderate (ventral and lateral nuclei of the trapezoid body) to faint (superior paraolivary nucleus). VGLUT2-ir was moderate to strong throughout the SOC, whereas VGLUT3 was only weakly expressed. These results extend previous reports on co-localization of VGLUTs in the auditory brainstem. As in the adult, specific features were seen during development for all three transporters. Intensity increases and decreases occurred with both VGLUT1 and VGLUT3, whereas VGLUT2-ir remained moderately high throughout development. A striking result was obtained with VGLUT3, which was only transiently expressed in the different SOC nuclei between P0 and P12. A transient occurrence of VGLUT1-immunoreactive terminals on somata of MSO neurons was another striking finding. Our results imply a considerable amount of synaptic reorganization in the glutamatergic inputs to the SOC and suggest differential roles of VGLUTs during maturation and in adulthood. This work was supported by the Graduate Research School Molecular, physiological and pharmacological analysis of cellular membrane transport, DFG GRK 845/1.     

Ultrastructure of an excitatory synapse

Summary The ultrastructure of the afferent synapse in hair cells of the lateral line-canal organ was studied using different fixation and staining techniques. Glutaraldehyde-fixed tissue without post-osmication, contrasted by section staining with uranyl acetate and lead citrate, was compared with (a) osmium tetroxide-fixed tissue followed by the same staining procedure, and with (b) glutaraldehyde-fixed tissue, block-impregnated with phosphotungstic acid (PTA). The results reveal a pronounced heterogeneity in the composition of the synaptic body, reflecting regional differences in chemical affinity to the fixatives and staining agents. It is proposed that the intracleft substance, the synaptic structure defined by the PTA staining technique, is actually due to the glutaraldehyde fixation procedure and is apparently the outer leaflet of the postsynaptic membrane. A special technique that allows alternate sections of a series to be differentially stained for electron microscopy is proposed.This research was carried out at the King Gustaf V Research Institute, Stockholm, Sweden     

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.     

Regulation of vesicular neurotransmitter transporters

Neurotransmitters are key molecules of neurotransmission. They are concentrated first in the cytosol and then in small synaptic vesicles of presynaptic terminals by the activity of specific neurotransmitter transporters of the plasma and the vesicular membrane, respectively. It has been shown that postsynaptic responses to single neurotransmitter packets vary over a wide range, which may be due to a regulation of vesicular neurotransmitter filling. Vesicular filling depends on the availability of transmitter molecules in the cytoplasm and the active transport into secretory vesicles relying on a proton gradient. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Go2 and Gq, which regulate VMAT activities in brain and platelets, respectively, and may also be involved in the regulation of VGLUTs. It appears that the vesicular content activates the G protein, suggesting a signal transduction form the luminal site which might be mediated by a vesicular G-protein coupled receptor or, as an alternative, possibly by the transporter itself. These novel functions of G proteins in the control of transmitter storage may link regulation of the vesicular content to intracellular signal cascades.     

Caenorhabditis elegans glutamate transporters influence synaptic function and behavior at sites distant from the synapse

To ensure precise neurotransmission and prevent neurotoxic accumulation, l-glutamate (Glu), the major excitatory neurotransmitter in the brain, is cleared from the synapse by glutamate transporters (GluTs). The molecular components of Glu synapses are highly conserved between Caenorhabditis elegans and mammals, yet the absence of synaptic insulation in C. elegans raises fundamental questions about Glu clearance strategies in the nematode nervous system. To gain insight into how Glu clearance is accomplished and how GluTs impact neurotransmission, we probed expression and function of all 6 GluTs found in the C. elegans genome. Disruption of each GluT impacts multiple Glu-dependent behaviors, with GluT combinations commonly increasing the severity of behavioral deficits. Interestingly, the sole GluT that we find expressed in neurons is localized predominantly in presynaptic neurons, in contrast to the postsynaptic concentration of neuronal GluTs typical in mammals. Moreover, 3 of the 6 GluT genes appear strongly expressed on the capillary excretory canal cell, where they affect Glu-dependent behaviors from positions distal to glutamatergic circuits. Indeed, our focused study of GLT-3, one of the distally expressed GluTs, shows that despite this distance, GLT-3 function can balance the activity mediated by synaptic release and synaptic receptors. The effects of distal GluTs on glutamatergic circuits support that Glu diffusion outside the vicinity of the synapse is a critical factor in C. elegans neurotransmission. Together with the presynaptic localization of neuronal GluTs, these observations suggest an unusual strategy for Glu clearance in C. elegans.     

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.     

Sequence and hydropathy profile analysis of two classes of secondary transporters

A structural class in the MemGen classification of membrane proteins is a set of evolutionary related proteins sharing a similar global fold. A structural class contains both closely related pairs of proteins for which homology is clear from sequence comparison and very distantly related pairs, for which it is not possible to establish homology based on sequence similarity alone. In the latter case the evolutionary link is based on hydropathy profile analysis. Here, we use these evolutionary related sets of proteins to analyze the relationship between E-values in BLAST searches, sequence similarities in multiple sequence alignments and structural similarities in hydropathy profile analyses. Two structural classes of secondary transporters termed ST[3], which includes the Ion Transporter (IT) superfamily and ST[4], which includes the DAACS family (TC# 2.A.23) were extracted from the NCBI protein database. ST[3] contains 2051 unique sequences distributed over 32 families and 59 subfamilies. ST[4] is a smaller class containing 399 unique sequences distributed over 2 families and 7 subfamilies. One subfamily in ST[4] contains a new class of binding protein dependent secondary transporters. Comparison of the averaged hydropathy profiles of the subfamilies in ST[3] and ST[4] revealed that the two classes represent different folds. Divergence of the sequences in ST[4] is much smaller than observed in ST[3], suggesting different constraints on the proteins during evolution. Analysis of the correlation between the evolutionary relationship of pairs of proteins in a class and the BLAST E-value revealed that: (i) the BLAST algorithm is unable to pick up the majority of the links between proteins in structural class ST[3], (ii) "low complexity filtering" and "composition based statistics" improve the specificity, but strongly reduce the sensitivity of BLAST searches for distantly related proteins, indicating that these filters are too stringent for the proteins analyzed, and (iii) the E-value cut-off, which may be used to evaluate evolutionary significance of a hit in a BLAST search is very different for the two structural classes of membrane proteins.     

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice

We assessed baseline and KCl-stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle-aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild-type littermates. In addition, we assessed the age-related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17-month-old APdE9 mice as compared to 7-month-old APdE9 mice. This age-dependent decrease in stimulated glutamate release was also evident in wild-type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild-type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age-dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT-1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle-aged APdE9 mice. Blunted pre-synaptic glutamate response may contribute to memory deficit in middle-aged APdE9 mice.     

Cryptic expression of functional glutamate transporters in the developing rodent brain

The co-ordinate functioning of neurons and glia is required for glutamate-mediated neurotransmission. In this study, we show by immunocytochemical detection of D-aspartate uptake, that functional glutamate transporters are present in the developing CNS of fetal and neonatal rats, including forebrain, midbrain and hindbrain, at least as early as embryonic day 12 (E12). Use of the transport inhibitor dihydrokainic acid revealed a significant role for GLT-1 in the uptake process.Immunolabelling for the glutamate transporters GLAST, GLT-1alpha and GLT-1v showed that each of these proteins are expressed early in development and appear to be restricted to glial-like cells throughout the development period examined (except in the retina, where neuronal elements were also labelled). Our capacity to detect very early expression of the variant forms of GLT-1 contrasts with other studies, a feature that we attribute to the use of antigen-recovery techniques that unmask protein epitopes that are otherwise undetectable. These studies illustrate the widespread presence of functional glutamate transporters in the developing CNS, in many cases before the onset of periods of synaptogenesis and indicate that regulation of extracellular glutamate by multiple excitatory amino acid transporters might be crucial in early CNS development.     

The structure of glutamate transporters shows channel-like features

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large family of secondary transporters, which includes transporters from a variety of bacterial, archaeal and eukaryotic organisms. The transporters consist of eight membrane-spanning alpha-helices and two pore-loop structures, which are unique among secondary transporters but may resemble pore-loops found in ion channels. Another distinctive structural feature is the presence of a highly amphipathic membrane-spanning alpha-helix that provides a hydrophilic path through the membrane. The unusual structural features of the transporters are discussed in relation to their function.     

Developmental pattern of three vesicular glutamate transporters in the myenteric plexus of the human fetal small intestine

Three vesicular glutamate transporters (VGLUT1-3) have previously been identified in the central nervous system, where they define the glutamatergic phenotype, and their expression is tightly regulated during brain development. In the present study we applied immunocytochemistry to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Immunoreactivity against all three VGLUTs was found predominantly in the ganglionic neuropil, interganglionic varicose fibers and perisomatic puncta, but cytoplasmic labeling with different intensities also occurred. Each transporter displayed a characteristic spatiotemporal expression pattern, with the transient increase or decrease of immunoreactive cell bodies, varicosities or perisomatic puncta, depending on the fetal age, the gut segment or the ganglionic compartment. Throughout gestational weeks 14-23, VGLUT1 immunoreactivity always predominated over VGLUT2 immunoreactivity, though both peaked around week 20. VGLUT3 immunoreactivity was less abundant in the developing myenteric plexus than those of VGLUT1 and VGLUT2 immunoreactivity. It was mainly expressed in the ganglionic neuropil and in the perisomatic puncta throughout the examined gestational period. Neuronal perikarya immunoreactive for VGLUT3 were restricted to between weeks 18 and 20 of gestation and exclusively to the oral part of the small intestine.     

Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.