Expression of glutamate and inhibitory amino acid vesicular transporters in the rodent auditory brainstem

Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus, suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 coexpressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits.     

Distributions of vesicular glutamate transporters 1 and 2 in the visual system of tree shrews (Tupaia belangeri)

Vesicular glutamate transporter (VGLUT) proteins regulate the storage and release of glutamate from synapses of excitatory neurons. Two isoforms, VGLUT1 and VGLUT2, are found in most glutamatergic projections across the mammalian visual system, and appear to differentially identify subsets of excitatory projections between visual structures. To expand current knowledge on the distribution of VGLUT isoforms in highly visual mammals, we examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the lateral geniculate nucleus (LGN), superior colliculus, pulvinar complex, and primary visual cortex (V1) in tree shrews (Tupaia belangeri), which are closely related to primates but classified as a separate order (Scandentia). We found that VGLUT1 was distributed in intrinsic and corticothalamic connections, whereas VGLUT2 was predominantly distributed in subcortical and thalamocortical connections. VGLUT1 and VGLUT2 were coexpressed in the LGN and in the pulvinar complex, as well as in restricted layers of V1, suggesting a greater heterogeneity in the range of efferent glutamatergic projections from these structures. These findings provide further evidence that VGLUT1 and VGLUT2 identify distinct populations of excitatory neurons in visual brain structures across mammals. Observed variations in individual projections may highlight the evolution of these connections through the mammalian lineage. J. Comp. Neurol. 523:1792–1808, 2015. © 2015 Wiley Periodicals, Inc.     

In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice

The vesicular glutamate transporter VGLUT1 loads synaptic vesicles with the neurotransmitter glutamate and thereby determines glutamate release at many synapses in the mammalian brain. Due to its function and selective localization, VGLUT1 is one of the most specific markers for glutamatergic synaptic vesicles. It has been used widely to identify glutamatergic synapses, and its expression levels are tightly correlated with changes in quantal size, modulations of synaptic plasticity, and corresponding behaviors. We generated a fluorescent VGLUT1(Venus) knock-in mouse for the analysis of VGLUT1 and glutamatergic synaptic vesicle trafficking. The mutation does not affect glutamatergic synapse function, and thus the new mouse model represents a universal tool for the analysis of glutamatergic transmitter systems in the forebrain. Previous studies demonstrated synaptic vesicle exchange between terminals in vitro. Using the VGLUT1(Venus) knock-in, we show that synaptic vesicles are dynamically shared among boutons in the cortex of mice in vivo. We provide a detailed analysis of synaptic vesicle sharing in vitro, and show that network homeostasis leads to dynamic scaling of synaptic VGLUT1 levels.     

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1)

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.     

Convergence of spinal trigeminal and cochlear nucleus projections in the inferior colliculus of the guinea pig

In addition to ascending auditory inputs, the external cortex of the inferior colliculus (ICX) receives prominent somatosensory inputs. To elucidate the extent of interaction between auditory and somatosensory representations at the level of IC, we explored the dual projections from the cochlear nucleus (CN) and the spinal trigeminal nucleus (Sp5) to the inferior colliculus (IC) in the guinea pig, using both retrograde and anterograde tracing techniques. Injections of retrograde tracers into ICX resulted in cell-labeling primarily in the contralateral DCN and pars interpolaris and caudalis of Sp5. Labeled cells in DCN were either fusiform or multipolar cells, whereas those in Sp5 varied in size and shape. Injections of anterograde tracers into either CN or Sp5 resulted in terminal labeling in ICX primarily on the contralateral side. Most projection fibers from Sp5 terminated in a laminar pattern from ventromedial to dorsolateral within the ventrolateral ICX, the ventral border of IC, and the ventromedial edge of IC (collectively termed "the ventrolateral border region of IC," ICXV). Less dense anterograde labeling was observed in lateral and rostral ICX. Injecting different tracers into both Sp5 and CN confirmed the overlapping areas of convergent projections from Sp5 and CN in IC: The most intense dual labeling was seen in the ICXV, and less intense dual labeling was also observed in the rostral part of ICX. This convergence of projection fibers from CN and Sp5 provides an anatomical substrate for multimodal integration in the IC.     

Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters

Three closely related proteins transport glutamate into synaptic vesicles for release by exocytosis. Complementary patterns of expression in glutamatergic terminals have been reported for VGLUT1 and VGLUT2. VGLUT3 shows expression by many cells not considered to be glutamatergic. Here we describe the changes in VGLUT expression that occur during development. VGLUT1 expression increases gradually after birth and eventually predominates over the other isoforms in telencephalic regions. Expressed at high levels shortly after birth, VGLUT2 declines with age in multiple regions, in the cerebellum by 14-fold. In contrast, Coexpression of the two isoforms occurs transiently during development as well as permanently in a restricted subset of glutamatergic terminals in the adult. VGLUT3 is transiently expressed at high levels by select neuronal populations, including terminals in the cerebellar nuclei, scattered neurons in the cortex, and progenitor-like cells, implicating exocytotic glutamate release in morphogenesis and development. VGLUT3 also colocalizes extensively during development with the neuronal vesicular monoamine transporter VMAT2, with the vesicular acetylcholine transporter VAChT, and with the vesicular gamma-aminobutyric acid transporter VGAT. Such coexpression occurs particularly at some specific developmental stages and is restricted to certain sets of cells. In skeletal muscle, VGLUT3 localizes to granular organelles in the axon terminal as well as in the muscle sarcoplasm. The results suggest novel mechanisms and roles for regulated transmitter release.     

Distribution of vesicular glutamate transporter mRNA in rat hypothalamus

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual-label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65-positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function.     

Hippocampal distribution of vesicular glutamate transporter 1 in patients with temporal lobe epilepsy

Purpose:   Vesicular glutamate transporters (VGLUTs) are responsible for loading synaptic vesicles with glutamate, determining the phenotype of glutamatergic neurons, and have been implicated in the regulation of quantal size and presynaptic plasticity. We analyzed VGLUT subtype expression in normal human hippocampus and tested the hypothesis that alterations in VGLUT expression may contribute to long-term changes in glutamatergic transmission reported in patients with temporal lobe epilepsy (TLE).
Methods:   VGLUT immunohistochemistry, immunofluorescence, in situ hybridization, Western blotting, and quantitative polymerase chain reaction (qPCR) were performed on biopsies from TLE patients without (non-HS) and with hippocampal sclerosis (HS) and compared to autopsy controls and rat hippocampus. VGLUT1 expression was compared with synaptophysin, neuropeptide Y (NPY), and Timm's staining.
Results:   VGLUT1 was the predominant VGLUT in human hippocampus and appeared to be localized to presynaptic glutamatergic terminals. In non-HS hippocampi, VGLUT1 protein levels were increased compared to control and HS hippocampi in all subfields. In HS hippocampi VGLUT1 expression was decreased in subfields with severe neuronal loss, but strongly up-regulated in the dentate gyrus, characterized by mossy fiber sprouting.
Discussion:   VGLUT1 is used as marker for glutamatergic synapses in the human hippocampus. In HS hippocampi VGLUT1 up-regulation in the dentate gyrus probably marks new glutamatergic synapses formed by mossy fiber sprouting. Our data indicate that non-HS patients have an increased capacity to store glutamate in vesicles, most likely due to an increase in translational processes or upregulation of VGLUT1 in synapses from afferent neurons outside the hippocampus. This up-regulation may increase glutamatergic transmission, and thus contribute to increased extracellular glutamate levels and hyperexcitability.     

Quantitative organization of neurotransmitters in the deep cerebellar nuclei of the Lurcher mutant

The Lurcher mutant mouse is characterized by a primary selective loss of Purkinje cells, leading to the near total apoptotic death of these neurons. In contrast to the subsequent massive secondary degeneration of the granule cells and the inferior olivary neurons, only mild degeneration occurs in the deep cerebellar nuclei (DCN). However, it is not known to what extent the different populations of DCN neurons-glutamatergic principal projection neurons, gamma-aminobutyric acid (GABA)-ergic inferior olivary projection neurons, and glycinergic neurons-are affected in their neurotransmitter composition. To answer this question we studied the neurotransmitter contents (glutamate, GABA, and glycine) of DCN neurons and the size of synaptic boutons immunohistochemically on serial semithin sections in both Lurcher and wild-type mice. Applying the physical dissector counting method, our results confirmed the mild degeneration (a reduction by 20%) of large glutamatergic neurons and a more pronounced degeneration of GABAergic (by 42%) and glycinergic neurons (by 45%). On the other hand, an analysis of neurons colabeled for both GABA and glycine, revealed that this specific colabeling increased in the Lurcher mutant (by 40%). In addition, both the GABA-immunolabeled (IL) (by 56%) and the glycine-IL (by 45%) synaptic boutons showed an increase in diameter in the mutant. The density of these boutons showed a decrease of 30% each. In summary, the increase in the number of neurons colabeled for GABA and glycine, together with the increase in the size of the inhibitory synaptic boutons, could help in providing the minimum inhibition needed to maintain a residual "cerebellar" functionality in the Lurcher DCN.     

Descending projections from the inferior colliculus to the dorsal cochlear nucleus are excitatory

Ascending projections of the dorsal cochlear nucleus (DCN) target primarily the contralateral inferior colliculus (IC). In turn, the IC sends bilateral descending projections back to the DCN. We sought to determine the nature of these descending axons in order to infer circuit mechanisms of signal processing at one of the earliest stages of the central auditory pathway. An anterograde tracer was injected in the IC of CBA/Ca mice to reveal terminal characteristics of the descending axons. Retrograde tracer deposits were made in the DCN of CBA/Ca and transgenic GAD67–EGFP mice to investigate the cells giving rise to these projections. A multiunit best frequency was determined for each injection site. Brains were processed by using standard histologic methods for visualization and examined by fluorescent, brightfield, and electron microscopy. Descending projections from the IC were inferred to be excitatory because the cell bodies of retrogradely labeled neurons did not colabel with EGFP expression in neurons of GAD67–EGFP mice. Furthermore, additional experiments yielded no glycinergic or cholinergic positive cells in the IC, and descending projections to the DCN were colabeled with antibodies against VGluT2, a glutamate transporter. Anterogradely labeled endings in the DCN formed asymmetric postsynaptic densities, a feature of excitatory neurotransmission. These descending projections to the DCN from the IC were topographic and suggest a feedback pathway that could underlie a frequency‐specific enhancement of some acoustic signals and suppression of others. The involvement of this IC–DCN circuit is especially noteworthy when considering the gating of ascending signal streams for auditory processing. J. Comp. Neurol. 525:773–793, 2017. © 2016 Wiley Periodicals, Inc.     

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.     

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1-immunoreactivity (-IR); no VGLUT2-IR was present in any synapses of the developing or adult retina. VGLUT1-IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4-P5. Rod terminals showed VGLUT1-IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1-IR around P8, 2-3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6-P8, most did not show VGLUT1-IR until P8-P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL.     

Expression of the mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus

Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system.     

Distribution of vesicular glutamate transporter 2 in auditory and song control brain regions in the adult zebra finch (Taeniopygia guttata)

The songbird brain has a system of interconnected nuclei that are specialized for singing and song learning. Wada et al. (2004; J. Comp. Neurol. 476:44–64) found a unique distribution of the mRNAs for glutamate receptor subunits in the song control brain areas of songbirds. In conjunction with data from electrophysiological studies, these finding indicate a role for the glutamatergic neurons and circuits in the song system. This study examines vesicular glutamate transporter 2 (VGLUT2) mRNA and protein expression in the zebra finch brain, particularly in auditory areas and song nuclei. In situ hybridization assays for VGLUT2 mRNA revealed high levels of expression in the ascending auditory nuclei (magnocellular, angular, and laminar nuclei; dorsal part of the lateral mesencephalic nucleus; ovoidal nucleus), high or moderate levels of expression in the telencephalic auditory areas (cudomedial mesopallium, field L, caudomedial nidopallium), and expression in the song nuclei (HVC, lateral magnocellular nucleus of the anterior nidopallium, robust nucleus of the arcopallium), where levels of expression were greater than in the surrounding brain subdivisions. Area X did not show expression of VGLUT2 mRNA. Nuclei in the descending motor pathway (dorsomedial nucleus of the intercollicular complex, retroambigual nucleus, tracheosyringeal motor nucleus of the hypoglossal nerve) expressed VGLUT2 mRNA. The target nuclei of VGLUT2 mRNA‐expressing nuclei showed immunoreactivity for VGLUT2 as well as hybridization signals for the mRNA of glutamate receptor subunits. The present findings demonstrate the origins and targets of glutamatergic neurons and indicate a central role for glutamatergic circuits in the auditory and song systems in songbirds. J. Comp. Neurol. 522:2129–2151, 2014. © 2013 Wiley Periodicals, Inc.     

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-α-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.     

Morphology and electrophysiological properties of hamster spinal dorsal horn neurons that express VGLUT2 and enkephalin

The excitatory amino acid glutamate mediates transmission at spinal synapses, including those formed by sensory afferent fibers and by intrinsic interneurons. The identity and physiological properties of glutamatergic dorsal horn neurons are poorly characterized despite their importance in spinal sensory circuits. Moreover, many intrinsic spinal glutamatergic synapses colocalize the opioid peptide enkephalin (ENK), but the neurons to which they belong are yet to be identified. Therefore, we used immunohistochemistry and confocal microscopy to investigate expression of the VGLUT2 vesicular glutamate transporter, an isoform reported in nonprimary afferent spinal synapses, and ENK in electrophysiologically identified neurons of hamster spinal dorsal horn. VGLUT2 immunoreactivity was localized in restricted fashion to axon varicosities of neurons recorded from laminae II-V, although the occurrence of immunolabeling in individual varicosities varied widely between cells (39 +/- 36%, n = 31 neurons). ENK colocalized with VGLUT2 in up to 77% of varicosities (17 +/- 21%, n = 21 neurons). The majority of neurons expressing VGLUT2 and/or ENK had axons with dense local terminations or projections consistent with propriospinal functions. VGLUT2 and ENK labeling were not correlated with cellular morphology, intrinsic membrane properties, firing patterns, or synaptic responses to sensory afferent stimulation. However, VGLUT2 expression was significantly higher in neurons with depolarized resting membrane potential. The results are new evidence for a population of dual-function dorsal horn interneurons that might provide another mechanism for limiting excitation within dorsal horn circuits during periods of strong sensory activation.