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Vesicular Glutamate Transporter (vesicular + glutamate_transporter)
Selected AbstractsLocalization of Vesicular Glutamate Transporter 2 mRNA in the Dorsal Root Ganglion of the Pigeon (Columba Livia)ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2009Y. Atoji Summary Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA-expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord. [source] Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2005Kouichi Nakamura Abstract Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single- and double-immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2-loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal-like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate-putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2-loaded glutamatergic axons maturate earlier than VGluT1-laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood. J. Comp. Neurol. 492:263,288, 2005. © 2005 Wiley-Liss, Inc. [source] Synapse-specific localization of vesicular glutamate transporters in the rat olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007Marie-Madeleine Gabellec Abstract Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have so far been identified, with distinct expression patterns in the adult brain. Here, we investigated the spatial distribution of the three VGLUTs in the rat olfactory bulb, a brain region containing a variety of glutamate synapses, both axodendritic and dendrodendritic. Using multilabelling confocal microscopy and electron microscopic immunocytochemistry, we showed that each VGLUT isoform has a highly selective localization in olfactory bulb synapses. VGLUT1 is present at dendrodendritic synapses established by the output neurones (mitral and tufted cells) with bulbar interneurones in the glomerular layer and external plexiform layer, as well as in axonal synapses of the granule cell layer. By contrast, VGLUT2 is strongly expressed in axon terminals of olfactory sensory neurones, which establish synapses with second-order neurones in the glomerular neuropil. VGLUT2 is also found in the outer part of the external plexiform layer and in the granule cell layer but colocalizes only partially with VGLUT1. Finally, we showed that VGLUT3 is exclusively located in the glomerular neuropil, where it colocalizes extensively with the vesicular inhibitory amino acid transporter vesicular GABA transporter, suggesting that it is associated with a subset of inhibitory synapses. Together, these observations extend previous findings on VGLUT distribution in the forebrain, and suggest that each VGLUT subtype has a specific function in the distinct features of axodendritic and dendrodendritic synapses that characterize the olfactory bulb circuit. [source] Presynaptic localization of an AMPA-type glutamate receptor in corticostriatal and thalamostriatal axon terminalsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2004Fumino Fujiyama Abstract The neostriatum is known to receive glutamatergic projections from the cerebral cortex and thalamic nuclei. Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) are located on axon terminals of corticostriatal and thalamostriatal afferents, respectively, whereas VGluT3 is found in axon terminals of cholinergic interneurons in the neostriatum. In the present study, the postsynaptic localization of ionotropic glutamate receptors was examined in rat neostriatum by the postembedding immunogold method for double labelling of VGluT and glutamate receptors. Immunoreactive gold particles for AMPA receptor subunits GluR1 and GluR2/3 were frequently found not only on postsynaptic but also on presynaptic profiles immunopositive for VGluT1 and VGluT2 in the neostriatum, and GluR4-immunoreactive particles were observed on postsynaptic and presynaptic profiles positive for VGluT1. Quantitative analysis revealed that 27,45% of GluR1-, GluR2-, GluR2/3- and GluR4-immunopositive particles found in VGluT1- or VGluT2-positive synaptic structures in the neostriatum were associated with the presynaptic profiles of VGluT-positive axons. In contrast, VGluT-positive presynaptic profiles in the neostriatum showed almost no immunoreactivity for NMDA receptor subunits NR1 or NR2A/B. Furthermore, almost no GluR2/3-immunopositive particles were observed in presynaptic profiles of VGluT3-positive (cholinergic) terminals that made asymmetric synapses in the neostriatum, or in those of VGluT1- or VGluT2-positive terminals in the neocortex. The present results indicate that AMPA receptor subunits but not NMDA receptor subunits are located on axon terminals of corticostriatal and thalamostriatal afferents, and suggest that glutamate released from these axon terminals controls the activity of the terminals through the presynaptic AMPA autoreceptors. [source] Cocaine- and amphetamine-regulated transcript peptide (CART) is present in peptidergic C primary afferents and axons of excitatory interneurons with a possible role in nociception in the superficial laminae of the rat spinal cordEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Márk Kozsurek Abstract Cocaine- and amphetamine-regulated transcript peptides (CART) have been implicated in the regulation of several physiological functions, including pain transmission. A dense plexus of CART-immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. In this study, we used antibody against CART peptide, together with markers for various types of primary afferents, interneurons and descending systems to determine the origin of the CART-immunoreactive axons in the superficial laminae of the rat spinal cord. Calcitonin gene-related peptide (CGRP), a marker for peptidergic primary afferents in the dorsal horn, was present in 72.6% and 34.8% of CART-immunoreactive axons in lamina I and II, respectively. The majority of these fibres also contained substance P (SP), while a few were somatostatin (SOM)-positive. The other subpopulation of CART-immunoreactive boutons in lamina I and II also expressed SP and/or SOM without CGRP, but contained vesicular glutamate transporter 2, which is present mainly in excitatory interneuronal terminals. Our data demonstrate that the majority of CART-immunoreactive axons in the spinal dorsal horn originate from peptidergic nociceptive primary afferents, while the rest arise from excitatory interneurons that contain SP or SOM. This strongly suggests that CART peptide can affect glutamatergic neurotransmission as well as the release and effects of SP and SOM in nociception and other sensory processes. [source] Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2006Fumino Fujiyama Abstract The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals immunopositive for vesicular glutamate transporter (VGluT) 2 were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. By contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both compartments. As axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment. [source] Evidence for vesicular glutamate transporter synapses onto gonadotropin-releasing hormone and other neurons in the rat medial preoptic areaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003J. Kiss Abstract The medial preoptic area is a key structure in the control of reproduction. Several data suggest that excitatory amino acids are involved in the regulation of this function and the major site of this action is the medial preoptic region. Data concerning the neuromorphology of the glutamatergic innervation of the medial preoptic area are fragmentary. The present investigations were focused on: (i) the morphology of the vesicular glutamate transporter 1 (VGluT1)- and vesicular glutamate transporter 2 (VGluT2)-immunoreactive nerve terminals, which are considered to be specific to presumed glutamatergic neuronal elements, in the medial preoptic area of rat; and (ii) the relationship between these glutamate transporter-positive endings and the gonadotropin-releasing hormone (GnRH) neurons in the region. Single- and double-label immunocytochemistry was used at the light and electron microscopic level. There was a weak to moderate density of VGluT1- and a moderate to intense density of VGluT2-immunoreactive elements in the medial preoptic area. Electron microscopy revealed that both VGluT1- and VGluT2-immunoreactive boutons made asymmetric type synaptic contacts with unlabelled neurons. VGluT2-labelled, but not VGluT1-labelled, axon terminals established asymmetric synaptic contacts on GnRH-immunostained neurons, mainly on their dendrites. The present findings are the first electron microscopic examinations on the glutamatergic innervation of the rat medial preoptic area. They provide direct neuromorphological evidence for the existence of direct glutamatergic innervation of GnRH and other neurons in the rat medial preoptic area. [source] The subcellular localization of GABAB receptor subunits in the rat substantia nigraEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Justin Boyes Abstract The inhibitory effects of GABA within the substantia nigra (SN) are mediated in part by metabotropic GABAB receptors. To better understand the mechanisms underlying these effects, we have examined the subcellular localization of the GABAB receptor subunits, GABAB1 and GABAB2, in SN neurons and afferents using pre-embedding immunocytochemistry combined with anterograde or retrograde labelling. In both the SN pars compacta (SNc) and pars reticulata (SNr), GABAB1 and GABAB2 showed overlapping, but distinct, patterns of immunolabelling. GABAB1 was more strongly expressed by putative dopaminergic neurons in the SNc than by SNr projection neurons, whereas GABAB2 was mainly expressed in the neuropil of both regions. Immunogold labelling for GABAB1 and GABAB2 was localized in presynaptic and postsynaptic elements throughout the SN. The majority of labelling was intracellular or was associated with extrasynaptic sites on the plasma membrane. In addition, labelling for both subunits was found on the presynaptic and postsynaptic membranes at symmetric, putative GABAergic synapses, including those formed by anterogradely labelled striatonigral and pallidonigral terminals. Labelling was also observed on the presynaptic membrane and at the edge of the postsynaptic density at asymmetric, putative excitatory synapses. Double immunolabelling, using the vesicular glutamate transporter 2, revealed the glutamatergic nature of many of the immunogold-labelled asymmetric synapses. The widespread distribution of GABAB subunits in the SNc and SNr suggests that GABAB -mediated effects in these regions are likely to be more complex than previously described, involving presynaptic autoreceptors and heteroreceptors, and postsynaptic receptors on different populations of SN neurons. [source] Cell type-dependent expression of HCN1 in the main olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003Noémi B. Holderith Abstract In many brain regions, hyperpolarization-activated cationic currents (Ih) are involved in the generation of rhythmic activities, but the role of Ih in olfactory oscillations remains unclear. Knowledge of the cellular and subcellular distributions of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) subunits is necessary for understanding the role of Ih in olfactory network activities. Using light microscopic immunocytochemistry, we demonstrate strong HCN1 labelling of the glomerular layer and moderate staining of granule cell, internal and external plexiform layers of the rat main olfactory bulb. In the glomerular layer, among many unlabelled neurons, two distinct subpopulations of juxtaglomerular cells are labelled. Approximately 10% of the juxtaglomerular cells strongly express HCN1. These small diameter cells are immunoreactive for GABA and comprise a subpopulation of periglomerular cells. An additional subset of juxtaglomerular cells (, 1%) expresses low levels of HCN1. They are large in diameter, GABA immunonegative but immunopositive for vesicular glutamate transporter 2, characterizing them as external tufted cells. Quantitative immunogold localization revealed that the somatic plasma membranes of periglomerular cells contain approximately four times more HCN1 labelling than those of external tufted cells. Unlike in cortical pyramidal cells, immunogold density for HCN1 does not significantly differ in somatic and dendritic plasma membranes of external tufted cells, indicating that post-synaptic potentials arriving at proximal and distal dendrites are modulated by the same density of Ih. Our results demonstrate a cell type-dependent expression of HCN1 in the olfactory bulb and predict a differential contribution of distinct juxtaglomerular cell types to network oscillations. [source] The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal hornEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003A. J. Todd Abstract 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. [source] Nesfatin-1 Influences the Excitability of Paraventricular Nucleus NeuronesJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2008C. J. Price Nesfatin-1 is a newly-discovered satiety peptide found in several nuclei of the hypothalamus, including the paraventricular nucleus. To begin to understand the physiological mechanisms underlying these satiety-inducing actions, we examined the effects of nesfatin-1 on the excitability of neurones in the paraventricular nucleus. Whole-cell current-clamp recordings from rat paraventricular nucleus neurones showed nesfatin-1 to have either hyperpolarising or depolarising effects on the majority of neurones tested. Both types of response were observed in neurones irrespective of classification based on electrophysiological fingerprint (magnocellular, neuroendocrine or pre-autonomic) or molecular phenotype (vasopressin, oxytocin, corticotrophin-releasing hormone, thyrotrophin-releasing hormone or vesicular glutamate transporter), determined using single cell reverse transcription-poylmerase chain reaction. Consequently, we provide the first evidence that this peptide, which is produced in the paraventricular nucleus, has effects on the membrane potential of a large proportion of different subpopulations of neurones located in this nucleus, and therefore identify nesfatin-1 as a potentially important regulator of paraventricular nucleus output. [source] Quantitative analysis of pre- and postsynaptic sex differences in the nucleus accumbensTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 8 2010Paul M. Forlano Abstract The nucleus accumbens (NAc) plays a central role in motivation and reward. While there is ample evidence for sex differences in addiction-related behaviors, little is known about the neuroanatomical substrates that underlie these sexual dimorphisms. We investigated sex differences in synaptic connectivity of the NAc by evaluating pre- and postsynaptic measures in gonadally intact male and proestrous female rats. We used DiI labeling and confocal microscopy to measure dendritic spine density, spine head size, dendritic length, and branching of medium spiny neurons (MSNs) in the NAc, and quantitative immunofluorescence to measure glutamatergic innervation using pre- (vesicular glutamate transporter 1 and 2) and postsynaptic (postsynaptic density 95) markers, as well as dopaminergic innervation of the NAc. We also utilized electron microscopy to complement the above measures. Clear but subtle sex differences were identified, namely, in distal dendritic spine density and the proportion of large spines on MSNs, both of which are greater in females. Sex differences in spine density and spine head size are evident in both the core and shell subregions, but are stronger in the core. This study is the first demonstration of neuroanatomical sex differences in the NAc and provides evidence that structural differences in synaptic connectivity and glutamatergic input may contribute to behavioral sex differences in reward and addiction. J. Comp. Neurol. 518:1330,1348, 2010. © 2009 Wiley-Liss, Inc. [source] Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2005Kouichi Nakamura Abstract Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single- and double-immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2-loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal-like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate-putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2-loaded glutamatergic axons maturate earlier than VGluT1-laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood. J. Comp. Neurol. 492:263,288, 2005. © 2005 Wiley-Liss, Inc. [source] Distribution of prospective glutamatergic, glycinergic, and GABAergic neurons in embryonic and larval zebrafishTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 1 2004Shin-Ichi Higashijima Abstract Zebrafish are an excellent model for studies of the functional organization of neuronal circuits, but little is known regarding the transmitter phenotypes of the neurons in their nervous system. We examined the distribution in spinal cord and hindbrain of neurons expressing markers of transmitter phenotype, including the vesicular glutamate transporter (VGLUT) genes for glutamatergic neurons, the neuronal glycine transporter (GLYT2) for glycinergic neurons, and glutamic acid decarboxylase (GAD65/67) for GABAergic neurons. All three markers were expressed in a large domain in the dorsal two-thirds of spinal cord, with additional, more ventral expression domains for VGLUT2 and GAD/GABA. In the large dorsal domain, dual in situ staining showed that GLYT2 -positive cells were intermingled with VGLUT2 cells, with no dual-stained neurons. Many of the neurons in the dorsal expression domain that were positive for GABA markers at embryonic stages were also positive for GLYT2, suggesting that the cells might use both GABA and glycine, at least early in their development. The intermingling of neurons expressing inhibitory and excitatory markers in spinal cord contrasted markedly with the organization in hindbrain, where neurons expressing a particular marker were clustered together to form stripes that were visible running from rostral to caudal in horizontal sections and from dorsomedial to ventrolateral in cross sections. Dual labeling showed that the stripes of neurons labeled with one transmitter marker alternated with stripes of cells labeled for the other transmitter phenotypes. The differences in the distribution of excitatory and inhibitory neurons in spinal cord versus hindbrain may be tied to differences in their patterns of development and functional organization. J. Comp. Neurol. 480:1,18, 2004. © 2004 Wiley-Liss, Inc. [source] Neurotransmitter properties of spinal interneurons in embryonic and larval zebrafishTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 1 2004Shin-Ichi Higashijima Abstract Many classes of spinal interneurons in zebrafish have been described based on morphology, but their transmitter phenotypes are largely unknown. Here we combine back-filling or genetic labeling of spinal interneurons with in situ staining for markers of neurotransmitter phenotypes, including the vesicular glutamate transporter (VGLUT) genes for glutamatergic neurons, the neuronal glycine transporter (GLYT2) for glycinergic neurons, and glutamic acid decarboxylase (GAD) for GABAergic neurons. Neurons positive for VGLUT include the commissural CoPA, MCoD, UCoD, and some of the CoSA neurons. The CiD interneurons, which have ipsilateral descending axons, were also VGLUT -positive, as were the ventrally located VeMe interneurons, whose descending axonal trajectory has not been clearly revealed. Cells positive for GLYT2 include the commissural CoLAs as well as some of the CoBL and CoSA neurons. The CiA cells were the only GLYT2 -positive cells with an ipsilateral axon. Cells staining for GAD included, most notably, the dorsal longitudinal ascending (DoLA) and KA interneurons. Our approach allowed us to define the likely transmitter phenotypes of most of the known classes of spinal interneurons. These data provide a foundation for understanding the functional organization of the spinal networks in zebrafish. J. Comp. Neurol. 480:19,37, 2004. © 2004 Wiley-Liss, Inc. [source] Neurochemical characterization of extrinsic innervation of the guinea pig rectumTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2004Catharina Olsson Abstract The presence of markers for parasympathetic, sympathetic, and glutamatergic or peptidergic sensory innervation was investigated by using in vitro tracing with biotinamide, combined with immunohistochemistry, to characterise quantitatively extrinsic axons to myenteric ganglia of the guinea pig rectum. Of biotinamide-filled varicose axons, 3.6 ± 1.3% were immunoreactive for tyrosine hydroxylase (TH) and 16.0 ± 4.8% for vesicular acetylcholine transporter (VAChT). TH and vesicular monoamine transporter (VMAT1) showed high coexistence (83,100%), indicating that varicosities lacking TH immunoreactivity also lacked VMAT1. VAChT was detectable in 77% of choline acetyltransferase (ChAT)-immunoreactive varicosities. Calcitonin gene-related peptide (CGRP) was detected in 5.3 ± 1.6% of biotinamide-labeled varicosities, the vesicular glutamate transporter (VGluT) 1 in 2.8 ± 0.8%, and VGluT2 in 11.3 ± 4.2% of varicosities of extrinsic origin. Varicosities from the same axon showed consistent immunoreactivity. A novel type of nerve ending was identified, with branching, flattened lamellar endings, similar to the intraganglionic laminar endings (IGLEs) of the proximal gut. Rectal IGLEs were frequently immunoreactive for VGluT1 and VGluT2. Thus most varicose axons of extrinsic origin, which innervate rectal myenteric ganglia, lack detectable levels of immunoreactivity for TH, VMAT1, VAChT, ChAT, VGluT1/2, or CGRP, under conditions in which these markers are readily detectable in other axons. Although some unlabeled varicosities may belong to afferent axons that lack detectable CGRP or VGluT1/2 in the periphery, this suggests that a large proportion of axons do not release any of the major autonomic or sensory transmitters. We speculate that this may vary under particular circumstances, for example, inflammation or obstruction of the gut. J. Comp. Neurol. 470:357,371, 2004. © 2004 Wiley-Liss, Inc. [source] Cellular sources, targets and actions of constitutive nitric oxide in the magnocellular neurosecretory system of the ratTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Javier E. Stern Nitric oxide (NO) is a key activity-dependent modulator of the magnocellular neurosecretory system (MNS) during conditions of high hormonal demand. In addition, recent studies support the presence of a functional consitutive NO tone. The aim of this study was to identify the cellular sources, targets, signalling mechanisms and functional relevance of constitutive NO production within the supraoptic nucleus (SON). Direct visualization of intracellular NO, along with neuronal nitric oxide synthase (nNOS) and cGMP immunohistochemisty, was used to study the cellular sources and targets of NO within the SON, respectively. Our results support the presence of a strong NO basal tone within the SON, and indicate that vasopressin (VP) neurones constitute the major neuronal source and target of basal NO. NO induced-fluorescence and cGMP immunoreactivity (cGMPir) were also found in the glia and microvasculature of the SON, suggesting that they contribute as sources/targets of NO within the SON. cGMPir was also found in association with glutamic acid decarboxylase 67 (GAD67)- and vesicular glutamate transporter 2 (VGLUT2)-positive terminals. Glutamate, acting on NMDA and possibly AMPA receptors, was found to be an important neurotransmitter driving basal NO production within the SON. Finally, electrophysiological recordings obtained from SON neurones in a slice preparation indicated that constitutive NO efficiently restrains ongoing firing activity of these neurones. Furthermore, phasically active (putative VP) and continuously firing neurones appeared to be influenced by NO originating from different sources. The potential roles for basal NO as an autocrine signalling molecule, and one that bridges neuronal,glial,vascular interactions within the MNS are discussed. [source] Localization of Vesicular Glutamate Transporter 2 mRNA in the Dorsal Root Ganglion of the Pigeon (Columba Livia)ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2009Y. Atoji Summary Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA-expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord. [source] Quantification and characterization of GABA-ergic amacrine cells in the retina of GAD67-GFP knock-in miceACTA OPHTHALMOLOGICA, Issue 4 2008Christian Albrecht May Abstract. Purpose:, Although the presence of ,-aminobutyrate acid (GABA) in amacrine cells and its co-localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA-ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse. Methods:, Whole mounts of the retina were prepared and the GFP-positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3. Results:, Displaced GABA-ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 ± 170 cells/mm2. In the inner nuclear layer (INL), the density of amacrine cells was 8821 ± 448 cells/mm2 in the central region and 6825 ± 408 cells/mm2 in the peripheral region. GFP-positive amacrine cells co-localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co-localization was seen with antibodies against PV, TH, and VGluT 1-3. Conclusions:, This study presents the first quantitative data concerning the co-localization of GABA-ergic neurons in the mouse retina with various neuronal markers. [source] Synapse-specific localization of vesicular glutamate transporters in the rat olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007Marie-Madeleine Gabellec Abstract Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have so far been identified, with distinct expression patterns in the adult brain. Here, we investigated the spatial distribution of the three VGLUTs in the rat olfactory bulb, a brain region containing a variety of glutamate synapses, both axodendritic and dendrodendritic. Using multilabelling confocal microscopy and electron microscopic immunocytochemistry, we showed that each VGLUT isoform has a highly selective localization in olfactory bulb synapses. VGLUT1 is present at dendrodendritic synapses established by the output neurones (mitral and tufted cells) with bulbar interneurones in the glomerular layer and external plexiform layer, as well as in axonal synapses of the granule cell layer. By contrast, VGLUT2 is strongly expressed in axon terminals of olfactory sensory neurones, which establish synapses with second-order neurones in the glomerular neuropil. VGLUT2 is also found in the outer part of the external plexiform layer and in the granule cell layer but colocalizes only partially with VGLUT1. Finally, we showed that VGLUT3 is exclusively located in the glomerular neuropil, where it colocalizes extensively with the vesicular inhibitory amino acid transporter vesicular GABA transporter, suggesting that it is associated with a subset of inhibitory synapses. Together, these observations extend previous findings on VGLUT distribution in the forebrain, and suggest that each VGLUT subtype has a specific function in the distinct features of axodendritic and dendrodendritic synapses that characterize the olfactory bulb circuit. [source] Glutamatergic neurons are present in the rat ventral tegmental areaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Tsuyoshi Yamaguchi Abstract The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or ,-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co,expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic. [source] Two populations of glutamatergic axons in the rat dorsal raphe nucleus defined by the vesicular glutamate transporters 1 and 2EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Kathryn G. Commons Abstract Most glutamatergic neurons in the brain express one of two vesicular glutamate transporters, vGlut1 or vGlut2. Cortical glutamatergic neurons highly express vGlut1, whereas vGlut2 predominates in subcortical areas. In this study immunohistochemical detection of vGlut1 or vGlut2 was used in combination with tryptophan hydroxylase (TPH) to characterize glutamatergic innervation of the dorsal raphe nucleus (DRN) of the rat. Immunofluorescence labeling of both vGlut1 and vGlut2 was punctate and homogenously distributed throughout the DRN. Puncta labeled for vGlut2 appeared more numerous then those labeled for vGlut1. Ultrastructural analysis revealed axon terminals containing vGlut1 and vGlut2 formed asymmetric-type synapses 80% and 95% of the time, respectively. Postsynaptic targets of vGlut1- and vGlut2-containing axons differed in morphology. vGlut1-labeled axon terminals synapsed predominantly on small-caliber (distal) dendrites (42%, 46/110) or dendritic spines (46%, 50/110). In contrast, vGlut2-containing axons synapsed on larger caliber (proximal) dendritic shafts (> 0.5 µm diameter; 48%, 78/161). A fraction of both vGlut1- or vGlut2-labeled axons synapsed onto TPH-containing dendrites (14% and 34%, respectively). These observations reveal that different populations of glutamate-containing axons innervate selective dendritic domains of serotonergic and non-serotonergic neurons, suggesting they play different functional roles in modulating excitation within the DRN. [source] The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal hornEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003A. J. Todd Abstract 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. [source] Age-dependent enhancement of inhibitory synaptic transmission in CA1 pyramidal neurons via GluR5 kainate receptorsHIPPOCAMPUS, Issue 8 2009Changqing Xu Abstract Changes in hippocampal synaptic networks during aging may contribute to age-dependent compromise of cognitive functions such as learning and memory. Previous studies have demonstrated that GABAergic synaptic transmission exhibits age-dependent changes. To better understand such age-dependent changes of GABAergic synaptic inhibition, we performed whole-cell recordings from pyramidal cells in the CA1 area of acute hippocampal slices on aged (24,26 months old) and young (2,4 months old) Brown-Norway rats. We found that the frequency and amplitude of spontaneous inhibitory postsynaptic current (IPSCs) were significantly increased in aged rats, but the frequency and amplitude of mIPSCs were decreased. Furthermore, the regulation of GABAergic synaptic transmission by GluR5 containing kainate receptors was enhanced in aged rats, which was revealed by using LY382884 (a GluR5 kainate receptor antagonist) and ATPA (a GluR5 kainate receptor agonist). Moreover, we demonstrated that vesicular glutamate transporters are involved in the kainate receptor dependent regulation of sIPSCs. Taken together, these results suggest that GABAergic synaptic transmission is potentiated in aged rats, and GluR5 containing kainate receptors regulate the inhibitory synaptic transmission through endogenous glutamate. These alterations of GABAergic input with aging could contribute to age-dependent cognitive decline. © 2009 Wiley-Liss, Inc. [source] Docking and homology modeling explain inhibition of the human vesicular glutamate transportersPROTEIN SCIENCE, Issue 9 2007Jonas Almqvist Abstract As membrane transporter proteins, VGLUT1,3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1,3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data. [source] VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cordTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Ida J. Llewellyn-Smith Abstract Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro-Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2-immunoreactive axons and synapses. CTB-immunoreactive SPN and PPN received many close appositions from VGLUT2-immunoreactive axons. VGLUT2-immunoreactive synapses occurred on Fluoro-Gold-labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1-immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1-immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1-immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarke's column. These data indicate that SPN and PPN are densely innervated by VGLUT2-immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1-immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN. J. Comp. Neurol. 503:741,767, 2007. © 2007 Wiley-Liss, Inc. 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