The Glutamatergic Neurons in the Spinal Cord of the Sea Lamprey: An In Situ Hybridization and Immunohistochemical Study

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.     

Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.     

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.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C→T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca²⁺-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

VGLUT2 controls heat and punctuate hyperalgesia associated with nerve injury via TRPV1-Cre primary afferents

Nerve injury induces a state of prolonged thermal and mechanical hypersensitivity in the innervated area, causing distress in affected individuals. Nerve injury-induced hypersensitivity is partially due to increased activity and thereby sustained release of neurotransmitters from the injured fibers. Glutamate, a prominent neurotransmitter in primary afferents, plays a major role in development of hypersensitivity. Glutamate is packed in vesicles by vesicular glutamate transporters (VGLUTs) to enable controlled release upon depolarization. While a role for peripheral VGLUTs in nerve injury-induced pain is established, their contribution in specific peripheral neuronal populations is unresolved. We investigated the role of VGLUT2, expressed by transient receptor potential vanilloid (TRPV1) fibers, in nerve injury-induced hypersensitivity. Our data shows that removal of Vglut2 from Trpv1-Cre neurons using transgenic mice abolished both heat and punctuate hyperalgesia associated with nerve injury. In contrast, the development of cold hypersensitivity after nerve injury was unaltered. Here, we show that, VGLUT2-mediated glutamatergic transmission from Trpv1-Cre neurons selectively mediates heat and mechanical hypersensitivity associated with nerve injury. Our data clarifies the role of the Trpv1-Cre population and the dependence of VGLUT2-mediated glutamatergic transmission in nerve injury-induced hyperalgesia.     

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.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements

The sequential stepping of left and right limbs is a fundamental motor behavior that underlies walking movements. This relatively simple locomotor behavior is generated by the rhythmic activity of motor neurons under the control of spinal neural networks known as central pattern generators (CPGs) that comprise multiple interneuron cell types. Little, however, is known about the identity and contribution of defined interneuronal populations to mammalian locomotor behaviors. We show a discrete subset of commissural spinal interneurons, whose fate is controlled by the activity of the homeobox gene Dbx1, has a critical role in controlling the left-right alternation of motor neurons innervating hindlimb muscles. Dbx1 mutant mice lacking these ventral interneurons exhibit an increased incidence of cobursting between left and right flexor/extensor motor neurons during drug-induced locomotion. Together, these findings identify Dbx1-dependent interneurons as key components of the spinal locomotor circuits that control stepping movements in mammals.     

The endocannabinoid system controls key epileptogenic circuits in the hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.     

Glutamatergic chemical transmission: look! Here, there, and anywhere

Vesicular glutamate transporter (VGLUT) is responsible for the active transport of L-glutamate in synaptic vesicles and thus plays an essential role in the glutamatergic chemical transmission in the central nervous system. VGLUT comprises three isoforms, VGLUT1, 2, and 3, and is a potential marker for the glutamatergic phenotype. Recent studies indicated that VGLUT is also expressed in non-neuronal cells, and localized with various organelles such as synaptic-like microvesicles in the pineal gland, and hormone-containing secretory granules in endocrine cells. L-Glutamate is stored in these organelles, secreted upon various forms of stimulation, and then acts as a paracrine-like modulator. Thus, VGLUTs highlight a novel framework of glutamatergic signaling and reveal its diverse modes of action.     

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

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

Huntington’s Disease and Group I Metabotropic Glutamate Receptors

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by involuntary body movement, cognitive impairment and psychiatric disturbance. A polyglutamine expansion in the amino-terminal region of the huntingtin (htt) protein is the genetic cause of HD. Htt protein interacts with a wide variety of proteins, and htt mutation causes cell signaling alterations in various neurotransmitter systems, including dopaminergic, glutamatergic, and cannabinoid systems, as well as trophic factor systems. This review will overview recent findings concerning htt-promoted alterations in cell signaling that involve different neurotransmitters and trophic factor systems, especially involving mGluR1/5, as glutamate plays a crucial role in neuronal cell death. The neuronal cell death that takes place in the striatum and cortex of HD patients is the most important factor underlying HD progression. Metabotropic glutamate receptors (mGluR1 and mGluR5) have a very controversial role in neuronal cell death and it is not clear whether mGluR1/5 activation either protects or exacerbates neuronal death. Thus, understanding how mutant htt protein affects glutamatergic receptor signaling will be essential to further establish a role for glutamate receptors in HD and develop therapeutic strategies to treat HD.     

Emerging evidence for a similar role of glutamate receptors in the nervous and immune systems

The role of glutamate receptors in synaptic transmission and excitotoxicity in the nervous system is well established. Recent evidence has emerged that glutamatergic mechanisms also exist in a wide variety of non-neuronal cells. In the case of thymocytes and lymphocytes, several types of glutamate receptor are expressed which can induce functional changes. This review focuses on the cellular function of NMDA-activated ionotropic and groups I and III metabotropic glutamate receptors in lymphocytes. Levels of exogenous and endogenous circulatory agonists and antagonists for lymphocyte glutamate receptors, notably homocysteine metabolites, are markedly increased in certain disease states and may be involved in disorders of the immune system. In addition to glutamate and aspartate, these compounds are active at glutamate receptors and increase the excitotoxic effects of glutamate in both neurons and lymphocytes. Increased levels of compounds acting at glutamate receptors may be risk factors for organ damage, for example in both heart and kidney disease. We conclude that glutamate is involved in signaling in immunocompetent cells and that the expression of both ionotropic and metabotropic glutamate receptors may have regulatory functions in immunocompetent cells, as well as in the nervous system. In addition, glutamate may serve as a signaling agent between the immune and nervous systems.     

Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors

The vanilloid receptor VR1 (TRPV1) is a temperature- and capsaicin-sensitive cation channel expressed by a class of primary afferents involved in nociception. To confirm the hypothesis that VR1-positive primary afferents are glutamatergic and contact spinal neurons that express the main classes of ionotropic glutamate receptors, we performed multiple immunofluorescent staining for VR1 and the glutamate transporter VGLUT2 (a specific marker for glutamatergic transmission) or AMPA and NMDA receptor subunits. VR1-positive cells in the dorsal root ganglion and boutons of their central afferent fibers in the dorsal horn expressed VGLUT2, and the latter contacted AMPA- or NMDA receptor-positive perikarya. Based on our previous observations of preferential targeting of VR1-positive primary afferents to spinal neurons that express the neurokinin receptor NK1 (Hwang et al., 2003), we further quantified the frequency of termination of VR1-positive afferents onto NK1-positive neurons co-expressing glutamate receptors. A larger fraction of NK1/NMDA receptors-positive than NK1/AMPA receptors-positive sites were contacted by VR1-positive boutons. We conclude that VR1-positive primary afferents in the rat use glutamate as neurotransmitter and contact postsynaptic sites that co-express NK1 and ionotropic glutamate receptors.     

Identification of Inhibitory Premotor Interneurons Activated at a Late Phase in a Motor Cycle during Drosophila Larval Locomotion

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs’ wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.     

Into great silence without VGLUT3

The vesicular glutamate transporters VGLUT1 and VGLUT2 fill synaptic vesicles with glutamate, an essential prerequisite for glutamatergic transmission in the CNS. In contrast, the third isoform, VGLUT3, is not confined to glutamatergic neurons, and its function has remained enigmatic. In this issue of Neuron, Seal et al. show that mice lacking VGLUT3 are profoundly deaf and exhibit nonconvulsive seizures.     

Neural circuit flexibility in a small sensorimotor system

Neuronal circuits underlying rhythmic behaviors (central pattern generators: CPGs) can generate rhythmic motor output without sensory input. However, sensory input is pivotal for generating behaviorally relevant CPG output. Here we discuss recent work in the decapod crustacean stomatogastric nervous system (STNS) identifying cellular and synaptic mechanisms whereby sensory inputs select particular motor outputs from CPG circuits. This includes several examples in which sensory neurons regulate the impact of descending projection neurons on CPG circuits. This level of analysis is possible in the STNS due to the relatively unique access to identified circuit, projection, and sensory neurons. These studies are also revealing additional degrees of freedom in sensorimotor integration that underlie the extensive flexibility intrinsic to rhythmic motor systems.