Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes.

The concept of a tripartite synapse including a presynaptic terminal, a postsynaptic spine, and an astrocytic process that responds to neuronal activity by fast gliotransmitter release, confers to the electrically silent astrocytes an active role in information processing. However, the mechanisms of gliotransmitter release are still highly controversial. The reported expression of all three vesicular glutamate transporters (VGLUT1-3) by astrocytes suggests that astrocytes, like neurons, may release glutamate by exocytosis. However, the demonstration of astrocytic VGLUT expression is largely based on immunostaining, and the possibility of nonspecific labeling needs to be systematically addressed. We therefore examined the expression of VGLUT1-3 in astrocytes, both in culture and in situ. We used Western blots and single-vesicle imaging by total internal reflection fluorescence microscopy in live cultured astrocytes, and confocal microscopy, at the cellular level in cortical, hippocampal, and cerebellar brain slices, combined with quantitative image analysis. Control experiments were systematically performed in cultured astrocytes using wild-type, VGLUT1-3 knock-out, VGLUT1(Venus) knock-in, and VGLUT2-EGFP transgenic mice. In fixed brain slices, we quantified the degree of overlap between VGLUT1-3 and neuronal or astrocytic markers, both in an object-based manner using fluorescence line profiles, and in a pixel-based manner using dual-color scatter plots followed by the calculation of Pearson's correlation coefficient over all pixels with intensities significantly different from background. Our data provide no evidence in favor of the expression of any of the three VGLUTs by gray matter protoplasmic astrocytes of the primary somatosensory cortex, the thalamic ventrobasal nucleus, the hippocampus, and the cerebellum.

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.

Vesicular glutamate transporters localization in the rat lingual papillae.

Vesicular glutamate transporters (VGluts) are widely expressed in neurons of the central nervous system, where they fulfil numerous functions during glutamatergic neurotransmission. This study examines their peripheral distribution in papillae of the tongue using immunohistochemical methods. VGlut 1 was detected in basal layers of the nontaste epithelium, and in intragemmal and perigemmal zones in taste papillae. Neither VGlut 2 nor VGlut 3 was detected in the lingual epithelium and taste papillae. These findings show the specific lingual pattern of distribution for VGluts and suggest that only VGlut 1 takes part in glutamatergic regulation of epithelial and taste cells within the tongue in physiological conditions.

Vesicular glutamate transporter mRNA expression in the medial temporal lobe in major depressive disorder, bipolar disorder, and schizophrenia.

BACKGROUND: Altered glutamate transmission has been found in the medial temporal lobe in severe psychiatric illnesses, including major depressive disorder (MDD) and bipolar disorder (BD). The vesicular glutamate transporters (VGLUTs) have a pivotal role in presynaptic release of glutamate into the synaptic cleft. We investigated this presynaptic marker in major psychiatric illness by measuring transcript expression of the VGLUTs in the medial temporal lobe. METHODS: The study sample comprised four groups of 13 subjects with MDD, BD, or schizophrenia (SCZ), and a comparison group from the Stanley Foundation Neuropathology Consortium. In situ hybridization was performed to quantify messenger RNA (mRNA) expression of VGLUT 1, 2, and 3 in medial temporal lobe structures. We also examined the same areas of rats treated with antidepressants, a mood stabilizer, and antipsychotics to assess the effects of these medications on VGLUT mRNA expression. RESULTS: We found decreased VGLUT1 mRNA expression in both MDD and BD in the entorhinal cortex (ERC), decreased VGLUT2 mRNA expression in MDD in the middle temporal gyrus, and increased VGLUT2 mRNA expression in SCZ in the inferior temporal gyrus (ITG). We also found a negative correlation between age and VGLUT1 mRNA expression in BD in the ERC and ITG. We did not find any changes in VGLUT mRNA expression in the hippocampus in any diagnostic group. We found decreased VGLUT1 mRNA expression in rats treated with haloperidol in the temporal cortex. CONCLUSIONS: These data indicate region-specific alterations of presynaptic glutamate innervation in the medial temporal lobe in the mood disorders.

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.

Localization of vesicular glutamate transporters in the peripheral vestibular system of rat.

OBJECTIVE: To examine the vesicular glutamate transporters (VGluTs: VGluT1-VGluT3) in the peripheral vestibular system. METHODS: The vestibular structures, including Scarpa's ganglion (vestibular ganglion, VG), maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs, by avidin-biotinylated peroxidase complex method, with 3-3'-diaminobenzidine (DAB) as chromogen. RESULTS: (1) VGluT1 was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs. (2) Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3-positive afferent fibers was in the maculae and ampullary cristae. (3) No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. CONCLUSION: These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.

Distribution of vesicular glutamate transporters in rat and human retina.

Central nervous system neurons have traditionally been thought to express exclusively membrane transporters and/or vesicular transporters for their transmitter. Three vesicular glutamate transporters have recently been cloned: BNPI/VGLUT1 (a brain-specific sodium-dependent inorganic phosphate (Pi) transporter), and its homologs DNPI/VGLUT2 (differentiation-associated sodium-dependent Pi transporter) and VGLUT3. We investigated the subcellular distributions of these three vesicular transporters in rat and human retina. VGLUT1 was present in the outer and inner plexiform layers (OPL and IPL), as shown by punctate staining in both human and rat retina. In the OPL, it was colocalized with synaptophysin, consistent with its expression in glutamatergic photoreceptor terminals, and it was present in PKC-alpha-labeled glutamatergic bipolar cell terminals in the IPL. By contrast, VGLUT2 was present in horizontal cells and ganglion cells in rat and human retina. In human retina, VGLUT2 was also found in some amacrine cells, including GAD-immunopositive amacrine cells. VGLUT3 was present in glycine-releasing amacrine cells in rat retina but was restricted to a few ganglion cells in human retina. The distribution of VGLUT1 in excitatory synaptic terminal was consistent with its involvement in glutamate release at excitatory synapses, whereas the cellular distributions of VGLUT2 and VGLUT3 suggested that these molecules may be involved in functions other than glutamate release, such as glutamate storage for GABA synthesis in non-glutamatergic neurons.

Galphao2 regulates vesicular glutamate transporter activity by changing its chloride dependence.

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.