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Fischer KD, Knackstedt LA, Rosenberg PA. Glutamate homeostasis and dopamine signaling: Implications for psychostimulant addiction behavior. Neurochem Int 2021; 144:104896. [PMID: 33159978 PMCID: PMC8489281 DOI: 10.1016/j.neuint.2020.104896] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Revised: 10/30/2020] [Accepted: 10/31/2020] [Indexed: 02/06/2023]
Abstract
Cocaine, amphetamine, and methamphetamine abuse disorders are serious worldwide health problems. To date, there are no FDA-approved medications for the treatment of these disorders. Elucidation of the biochemical underpinnings contributing to psychostimulant addiction is critical for the development of effective therapies. Excitatory signaling and glutamate homeostasis are well known pathophysiological substrates underlying addiction-related behaviors spanning multiple types of psychostimulants. To alleviate relapse behavior to psychostimulants, considerable interest has focused on GLT-1, the major glutamate transporter in the brain. While many brain regions are implicated in addiction behavior, this review focuses on two regions well known for their role in mediating the effects of cocaine and amphetamines, namely the nucleus accumbens (NAc) and the ventral tegmental area (VTA). In addition, because many investigators have utilized Cre-driver lines to selectively control gene expression in defined cell populations relevant for psychostimulant addiction, we discuss potential off-target effects of Cre-recombinase that should be considered in the design and interpretation of such experiments.
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Affiliation(s)
- Kathryn D Fischer
- Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
| | - Lori A Knackstedt
- Psychology Department, University of Florida, Gainesville, FL, 32611, USA
| | - Paul A Rosenberg
- Department of Neurology, F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, 02115, USA; Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA.
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Ormel L, Lauritzen KH, Schreiber R, Kunzelmann K, Gundersen V. GABA, but Not Bestrophin-1, Is Localized in Astroglial Processes in the Mouse Hippocampus and the Cerebellum. Front Mol Neurosci 2020; 13:135. [PMID: 32848599 PMCID: PMC7399226 DOI: 10.3389/fnmol.2020.00135] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Accepted: 07/07/2020] [Indexed: 11/13/2022] Open
Abstract
GABA is proposed to act as a gliotransmitter in the brain. Differences in GABA release from astroglia are thought to underlie differences in tonic inhibition between the cerebellum and the CA1 hippocampus. Here we used quantitative immunogold cytochemistry to localize and compare the levels of GABA in astroglia in these brain regions. We found that the density of GABA immunogold particles was similar in delicate processes of Bergman glia in the cerebellum and astrocytes in the CA1 hippocampus. The astrocytic GABA release is proposed to be mediated by, among others, the Ca2+ activated Cl- channel bestrophin-1. The bestrophin-1 antibodies did not show any significant bestrophin-1 signal in the brain of wt mice, nor in bestrophin-1 knockout mice. The bestrophin-1 signal was low both on Western blots and immunofluorescence laser scanning microscopic images. These results suggest that GABA is localized in astroglia, but in similar concentrations in the cerebellum and CA1 hippocampus, and thus cannot account for differences in tonic inhibition between these brain regions. Furthermore, our data seem to suggest that the GABA release from astroglia previously observed in the hippocampus and cerebellum occurs via mechanisms other than bestrophin-1.
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Affiliation(s)
- Lasse Ormel
- Section of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Department of Neurology, Oslo University Hospital, Ullevål, Oslo, Norway
| | - Knut H Lauritzen
- Section of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway
| | - Rainer Schreiber
- Department of Physiology, University of Regensburg, Regensburg, Germany
| | - Karl Kunzelmann
- Department of Physiology, University of Regensburg, Regensburg, Germany
| | - Vidar Gundersen
- Section of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.,Section for Movement Disorders, Department of Neurology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
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Parkin GM, Gibbons A, Udawela M, Dean B. Excitatory amino acid transporter (EAAT)1 and EAAT2 mRNA levels are altered in the prefrontal cortex of subjects with schizophrenia. J Psychiatr Res 2020; 123:151-158. [PMID: 32065951 DOI: 10.1016/j.jpsychires.2020.02.004] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2019] [Revised: 02/06/2020] [Accepted: 02/07/2020] [Indexed: 12/21/2022]
Abstract
Excitatory amino acid transporter (EAAT)1 and EAAT2 mediate glutamatergic neurotransmission and prevent excitotoxicity through binding and transportation of glutamate into glia. These EAATs may be regulated by metabotropic glutamate receptor 5 (mGluR5), which is also expressed by glia. Whilst we have data from an Affymetrix™ Human Exon 1.0 ST Array showing higher levels of EAAT1 mRNA (+36%) in Brodmann's are (BA)9 of subjects with schizophrenia, there is evidence that EAAT1 and EAAT2, as well as mGluR5 levels, are altered in the cortex of subjects with the disorder. Hence, we measured mRNA levels of these genes in other cortical regions in subjects with that disorder. EAAT1, EAAT2 and mGluR5 mRNA were measured, in triplicate, using Quantitative PCR in BA10 and BA46 from subjects with schizophrenia (n = 20) and age and sex matched controls (n = 18). Levels of mRNA were normalised to the geometric mean of two reference genes, transcription factor B1, mitochondrial (TFB1M) and S-phase kinase-associated protein 1A (SKP1A), for which mRNA did not vary between diagnostic groups in either region. Normalised levels of EAAT1 and EAAT2 mRNA were significantly higher in BA10 (EAAT1: U = 58, p = 0.0002; EAAT2 U = 70, p = 0.0009), but not BA46 (EAAT1: U = 122, p = 0.09; EAAT2: U = 136, p = 0.21), from subjects with schizophrenia compared to controls. mGluR5 levels in BA10 (U = 173, p=0.85) and BA46 (U = 178, p = 0.96) did not vary by cohort. Our data suggests that region-specific increases in cortical EAAT1 and EAAT2 mRNA are involved in schizophrenia pathophysiology and that disrupted glutamate uptake in schizophrenia may be of particular significance in BA10.
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Affiliation(s)
- Georgia M Parkin
- The Molecular Psychiatry Laboratory, The Florey Institute for Neuroscience and Mental Health, Parkville, Victoria, Australia; The Cooperative Research Centre for Mental Health, Parkville, Victoria, Australia.
| | - Andrew Gibbons
- The Molecular Psychiatry Laboratory, The Florey Institute for Neuroscience and Mental Health, Parkville, Victoria, Australia
| | - Madhara Udawela
- The Molecular Psychiatry Laboratory, The Florey Institute for Neuroscience and Mental Health, Parkville, Victoria, Australia; The Cooperative Research Centre for Mental Health, Parkville, Victoria, Australia
| | - Brian Dean
- The Molecular Psychiatry Laboratory, The Florey Institute for Neuroscience and Mental Health, Parkville, Victoria, Australia; The Cooperative Research Centre for Mental Health, Parkville, Victoria, Australia; The Centre for Mental Health, The Faculty of Health, Arts and Design, Swinburne University, Hawthorne, Victoria, Australia
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Parkin GM, Udawela M, Gibbons A, Dean B. Glutamate transporters, EAAT1 and EAAT2, are potentially important in the pathophysiology and treatment of schizophrenia and affective disorders. World J Psychiatry 2018; 8:51-63. [PMID: 29988908 PMCID: PMC6033743 DOI: 10.5498/wjp.v8.i2.51] [Citation(s) in RCA: 92] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2018] [Revised: 05/15/2018] [Accepted: 06/09/2018] [Indexed: 02/05/2023] Open
Abstract
Glutamate is the predominant excitatory neurotransmitter in the human brain and it has been shown that prolonged activation of the glutamatergic system leads to nerve damage and cell death. Following release from the pre-synaptic neuron and synaptic transmission, glutamate is either taken up into the pre-synaptic neuron or neighbouring glia by transmembrane glutamate transporters. Excitatory amino acid transporter (EAAT) 1 and EAAT2 are Na+-dependant glutamate transporters expressed predominantly in glia cells of the central nervous system. As the most abundant glutamate transporters, their primary role is to modulate levels of glutamatergic excitability and prevent spill over of glutamate beyond the synapse. This role is facilitated through the binding and transportation of glutamate into astrocytes and microglia. The function of EAAT1 and EAAT2 is heavily regulated at the levels of gene expression, post-transcriptional splicing, glycosylation states and cell-surface trafficking of the protein. Both glutamatergic dysfunction and glial dysfunction have been proposed to be involved in psychiatric disorder. This review will present an overview of the roles that EAAT1 and EAAT2 play in modulating glutamatergic activity in the human brain, and mount an argument that these two transporters could be involved in the aetiologies of schizophrenia and affective disorders as well as represent potential drug targets for novel therapies for those disorders.
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Affiliation(s)
- Georgia M Parkin
- Molecular Psychiatry Laboratory, the Florey Institute of Neuroscience and Mental Health, Parkville VIC 3052, Australia
- CRC for Mental Health, Carlton VIC 3053, Australia
| | - Madhara Udawela
- Molecular Psychiatry Laboratory, the Florey Institute of Neuroscience and Mental Health, Parkville VIC 3052, Australia
- CRC for Mental Health, Carlton VIC 3053, Australia
| | - Andrew Gibbons
- Molecular Psychiatry Laboratory, the Florey Institute of Neuroscience and Mental Health, Parkville VIC 3052, Australia
| | - Brian Dean
- Molecular Psychiatry Laboratory, the Florey Institute of Neuroscience and Mental Health, Parkville VIC 3052, Australia
- CRC for Mental Health, Carlton VIC 3053, Australia
- Research Centre for Mental Health, the Faculty of Health, Arts and Design, Swinburne University, Hawthorne VIC 3122, Australia
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Jolly S, Bazargani N, Quiroga AC, Pringle NP, Attwell D, Richardson WD, Li H. G protein-coupled receptor 37-like 1 modulates astrocyte glutamate transporters and neuronal NMDA receptors and is neuroprotective in ischemia. Glia 2017; 66:47-61. [PMID: 28795439 PMCID: PMC5724489 DOI: 10.1002/glia.23198] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2017] [Revised: 07/05/2017] [Accepted: 07/13/2017] [Indexed: 12/21/2022]
Abstract
We show that the G protein‐coupled receptor GPR37‐like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1–/– mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1‐mediated signalling inhibited astrocyte glutamate transporters and – surprisingly, given its lack of expression in neurons – reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D‐serine or TNF‐α, two astrocyte‐derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ∼40% in Gpr37l1–/– brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.
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Affiliation(s)
- Sarah Jolly
- Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, United Kingdom
| | - Narges Bazargani
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, WC1E 6BT, United Kingdom
| | - Alejandra C Quiroga
- Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, United Kingdom
| | - Nigel P Pringle
- Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, United Kingdom
| | - David Attwell
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, WC1E 6BT, United Kingdom
| | - William D Richardson
- Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, United Kingdom
| | - Huiliang Li
- Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, United Kingdom
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Fernández-López B, Valle-Maroto SM, Barreiro-Iglesias A, Rodicio MC. Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys. Glia 2014; 62:1254-69. [PMID: 24733772 DOI: 10.1002/glia.22678] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 03/13/2014] [Accepted: 04/03/2014] [Indexed: 12/27/2022]
Abstract
In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid-contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL-TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys.
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Affiliation(s)
- Blanca Fernández-López
- Department of Cell Biology and Ecology, CIBUS, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain
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Villar-Cerviño V, Barreiro-Iglesias A, Mazan S, Rodicio MC, Anadón R. Glutamatergic neuronal populations in the forebrain of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study. J Comp Neurol 2012; 519:1712-35. [PMID: 21452205 DOI: 10.1002/cne.22597] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Despite the importance of glutamate as a major excitatory neurotransmitter in the brain, the distribution of glutamatergic populations in the brain of most vertebrates is still unknown. Here, we studied for the first time the distribution of glutamatergic neurons in the forebrain of the sea lamprey (Petromyzon marinus), belonging to the most ancient group of vertebrates (agnathans). For this, we used in situ hybridization with probes for a lamprey vesicular glutamate transporter (VGLUT) in larvae and immunofluorescence with antiglutamate antibodies in both larvae and adults. We also compared glutamate and γ-aminobutyric acid (GABA) immunoreactivities in sections using double-immunofluorescence methods. VGLUT-expressing neurons were observed in the olfactory bulb, pallium, septum, subhippocampal lobe, preoptic region, thalamic eminence, prethalamus, thalamus, epithalamus, pretectum, hypothalamus, posterior tubercle, and nucleus of the medial longitudinal fascicle. Comparison of VGLUT signal and glutamate immunoreactivity in larval forebrain revealed a consistent distribution of positive cells, which were numerous in most regions. Glutamate-immunoreactive cell populations were also found in similar regions of the adult forebrain. These include mitral-like cells of the olfactory bulbs and abundant cells in the lateral pallium, septum, and various diencephalic regions, mainly in the prethalamus, thalamus, habenula, pineal complex, and pretectum. Only a small portion of the glutamate-immunoreactive cells showed colocalization with GABA, which was observed mainly in the olfactory bulb, telencephalon, hypothalamus, ventral thalamus, and pretectum. Comparison with glutamatergic cells observed in rodent forebrains suggests that the regional distribution of glutamatergic cells does not differ greatly in lampreys and mammals.
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Affiliation(s)
- Verona Villar-Cerviño
- Departamento de Biología Celular y Ecología, Facultad de Biología, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain
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Cooke RM, Luco S, Parker D. Manipulations of spinal cord excitability evoke developmentally-dependent compensatory changes in the lamprey spinal cord. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2012; 198:25-41. [DOI: 10.1007/s00359-011-0683-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Revised: 09/09/2011] [Accepted: 09/14/2011] [Indexed: 10/15/2022]
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Bergersen LH, Morland C, Ormel L, Rinholm JE, Larsson M, Wold JFH, Røe AT, Stranna A, Santello M, Bouvier D, Ottersen OP, Volterra A, Gundersen V. Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes. ACTA ACUST UNITED AC 2011; 22:1690-7. [PMID: 21914633 DOI: 10.1093/cercor/bhr254] [Citation(s) in RCA: 79] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.
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Affiliation(s)
- L H Bergersen
- Department of Anatomy, Centre for Molecular Biology and Neuroscience, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway
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A role for glutamate transporters in the regulation of insulin secretion. PLoS One 2011; 6:e22960. [PMID: 21853059 PMCID: PMC3154915 DOI: 10.1371/journal.pone.0022960] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2011] [Accepted: 07/02/2011] [Indexed: 11/19/2022] Open
Abstract
In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.
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D'Aniello S, Somorjai I, Garcia-Fernàndez J, Topo E, D'Aniello A. D-Aspartic acid is a novel endogenous neurotransmitter. FASEB J 2010; 25:1014-27. [PMID: 21163862 DOI: 10.1096/fj.10-168492] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
D-aspartic acid (D-Asp) is present in invertebrate and vertebrate neuroendocrine tissues, where it carries out important physiological functions and is implicated in nervous system development. We show here that D-Asp is a novel endogenous neurotransmitter in two distantly related animals, a mammal (Rattus norvegicus) and a mollusk (Loligo vulgaris). Our main findings demonstrate that D-Asp is present in high concentrations in the synaptic vesicles of axon terminals; synthesis for this amino acid occurs in neurons by conversion of L-Asp to D-Asp via D-aspartate racemase; depolarization of nerve endings with K(+) ions evokes an immediate release of D-Asp in a Ca(2+) dependent manner; specific receptors for D-Asp occur at the postsynaptic membrane, as demonstrated by binding assays and by the expansion of squid skin chromatophores; D-aspartate oxidase, the specific enzyme that oxidizes D-Asp, is present in the postsynaptic membranes; and stimulation of nerve endings with D-Asp triggers signal transduction by increasing the second messenger cAMP. Taken together, these data demonstrate that D-Asp fulfills all criteria necessary to be considered a novel endogenous neurotransmitter. Given its known role in neurogenesis, learning, and neuropathologies, our results have important implications for biomedical and clinical research.
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Affiliation(s)
- Salvatore D'Aniello
- Departament de Genètica, Institut de Biomedicina, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain
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Hoffman N, Parker D. Lesioning alters functional properties in isolated spinal cord hemisegmental networks. Neuroscience 2010; 168:732-43. [PMID: 20394805 DOI: 10.1016/j.neuroscience.2010.04.014] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2009] [Revised: 04/07/2010] [Accepted: 04/07/2010] [Indexed: 11/30/2022]
Abstract
Hemisegmental networks produced by longitudinal lesions of the spinal cord midline are able to generate rhythmic bursting activity. This has led to the suggestion that hemisegmental networks can independently burst in the intact spinal cord. Previous analyses in the lamprey spinal cord failed to show hemisegmental bursting in NMDA. This was subsequently attributed to the failure to wait sufficient time for NMDA-evoked hemisegmental activity to recover after being abolished by the lesion, which can take tens of minutes to hours. The reason for this delay in the onset of NMDA-evoked activity was not previously addressed. We have investigated it here by examining two hypotheses: that hemisegmental networks intrinsically burst under normal conditions but that NMDA-evoked bursting was temporarily silenced by lesion-induced transmitter release; or that lesioning altered functional properties in the hemisegment that subsequently led to the development of bursting. We found no evidence to support transmitter-induced silencing of ongoing NMDA-evoked hemisegmental activity, but did find evidence for significant changes in the cellular and synaptic properties of motor neurons and premotor excitatory interneurons in lesioned hemisegmental networks. These results thus suggest that there are lesion-induced changes in functional properties in hemisegmental networks. As the interpretation of lesion studies rests on the assumption that the functional properties of hemisegmental components are not altered, further work is needed before conclusions can be made about the function of the intact system.
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Affiliation(s)
- N Hoffman
- Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Site, Cambridge CB2 3DY, UK
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13
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d-Aspartate binding sites in rat Harderian gland. Amino Acids 2009; 38:229-35. [DOI: 10.1007/s00726-008-0231-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2008] [Accepted: 12/29/2008] [Indexed: 10/21/2022]
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Arranz AM, Hussein A, Alix JJP, Pérez-Cerdá F, Allcock N, Matute C, Fern R. Functional glutamate transport in rodent optic nerve axons and glia. Glia 2008; 56:1353-67. [PMID: 18551624 DOI: 10.1002/glia.20703] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na(+)-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake.
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Affiliation(s)
- Amaia M Arranz
- Departamento de Neurociencias, Universidad del País Vasco, Leioa, Spain
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Holten AT, Talgøy HA, Danbolt NC, Christian DN, Shimamoto K, Gundersen V, Vidar G. Low-affinity excitatory amino acid uptake in hippocampal astrocytes: a possible role of Na+/dicarboxylate cotransporters. Glia 2008; 56:990-7. [PMID: 18442087 DOI: 10.1002/glia.20672] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The excitatory amino acid transporters (EAATs) underlie the so-called "high affinity" uptake of glutamate, which is well characterized. In contrast, the "low-affinity" uptake of glutamate remains poorly defined, and it has been discussed whether it may represent a mere in vitro artifact. Here we have visualized "low-affinity" excitatory amino acid uptake sites by incubating rat hippocampal slices with the glutamate analogue D-aspartate in the presence of PMB-TBOA, which blocks the EAATs. After fixation of the slices, D-aspartate taken up into the tissue was localized with the use of light microscopic immunoperoxidase and electron microscopic immunogold methods, exploiting highly specific antibodies against D-aspartate. PMB-TBOA blocked uptake of both low and high exogenous D-aspartate concentrations (0.01-1.0 mM) into nerve terminals, as well as the uptake of 0.01 mM D-aspartate into astrocytes. Interestingly, there was a residual PMB-TBOA insensitive uptake of D-aspartate in astrocytes at higher exogenous D-aspartate concentrations (0.05-1.0 mM), strongly suggesting that astrocytes have "low-affinity" uptake sites for excitatory amino acid. The PMB-TBOA insensitive D-aspartate uptake in astrocytes was sodium dependent and inhibited by succinate and to certain extent by homocysteate, but not by cystine or DIDS. We suggest that excitatory amino acid is transported into astrocytes in a "low-affinity" fashion by sodium/dicarboxylate transporters.
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Glial-toxin-mediated disruption of spinal cord locomotor network function and its modulation by 5-HT. Neuroscience 2008; 153:1332-43. [PMID: 18440149 DOI: 10.1016/j.neuroscience.2008.03.034] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2007] [Revised: 03/18/2008] [Accepted: 03/19/2008] [Indexed: 11/22/2022]
Abstract
While it is established that glial cells actively influence neuronal and synaptic properties, the functional effects of glial-neuronal interactions are still not well understood. To address the role of glia at the network level we have examined the effects of the specific gliotoxin L-aminoadipic acid on the locomotor network output and cellular and synaptic properties in the lamprey spinal cord. The gliotoxic effect of aminoadipic acid was associated with a specific depolarization of glial cells. Aminoadipic acid depolarized the membrane potential of spinal cord neurons, suggesting a functional link between glia and neurons. The depolarization was significantly reduced by glutamate receptor antagonists in adults, but by gap junction blockers in larvae, suggesting a developmental difference in glial-neuronal interactions. Aminoadipic acid also reduced the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs), an effect that was not associated with changes in the presynaptic release probability or postsynaptic response to glutamate. These cellular and synaptic effects of aminoadipic acid were associated with disruption of the locomotor network output. This could not be accounted for by changes in glutamate uptake or potassium buffering by glia, suggesting a direct role for glia in the network. Interestingly, we found that the aminoadipic acid-evoked disruption of network activity and reduction of monosynaptic EPSP amplitudes did not occur in the presence of the endogenous spinal modulator 5-HT. These results thus provide evidence for an active functional role for glial cells in spinal cord locomotor networks, and suggest a potential glial modulatory effect of 5-HT.
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17
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Zielinski BS, Fredricks K, McDonald R, Zaidi AU. Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L. ACTA ACUST UNITED AC 2006; 34:209-16. [PMID: 16841164 DOI: 10.1007/s11068-005-8354-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2005] [Revised: 02/08/2006] [Accepted: 02/09/2006] [Indexed: 10/24/2022]
Abstract
This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.
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Affiliation(s)
- Barbara S Zielinski
- Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada, N9B 3P4.
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18
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Molchanova SM, Oja SS, Saransaari P. Taurine attenuates D-[3H]aspartate release evoked by depolarization in ischemic corticostriatal slices. Brain Res 2006; 1099:64-72. [PMID: 16781687 DOI: 10.1016/j.brainres.2006.04.105] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2005] [Revised: 04/23/2006] [Accepted: 04/28/2006] [Indexed: 02/06/2023]
Abstract
Taurine is thought to be protective in ischemia due to its neuroinhibitory effects. The present aim was to assess the ability of taurine to attenuate glutamate release evoked by ischemia and to determine which component of this release is affected. The release of preloaded D-[(3)H]aspartate (a non-metabolized analog of glutamate) from superfused murine corticostriatal slices was used as index of glutamate release. Preincubation of corticostriatal slices with 10 mM taurine reduced the D-[(3)H]aspartate release evoked by either chemical ischemia (0.5 mM NaCN in glucose-free medium) or oxygen-glucose deprivation. The taurine uptake inhibitor guanidinoethanesulfonate (5 mM), the glycine receptor antagonist strychnine (0.1 mM) and the GABA(A) receptor antagonist bicuculline (0.1 mM) did not block the taurine effect. To determine which component of ischemia-induced glutamate release is affected by taurine, three pathways of this release were pharmacologically modeled. Unlabeled D-aspartate (0.5 mM) and hypo-osmotic medium (NaCl reduced by 50 mM) evoked D-[(3)H]aspartate release via homoexchange and hypo-osmotic release pathways, respectively. Taurine did not influence these pathways. However, it suppressed the synaptic release of D-[(3)H]aspartate evoked by the voltage-gated sodium channel opener veratridine (0.1 mM). Taurine thus reduces glutamate release under ischemic conditions by affecting the depolarization-evoked component.
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19
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Gong XQ, Frandsen A, Lu WY, Wan Y, Zabek RL, Pickering DS, Bai D. D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons. Br J Pharmacol 2005; 145:449-59. [PMID: 15806114 PMCID: PMC1576155 DOI: 10.1038/sj.bjp.0706199] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
1 The amino acid, D-aspartate, exists in the mammalian brain and is an agonist at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D-aspartate on alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D-aspartate inhibited kainate-induced AMPAR current in hippocampal neurons. The inhibitory action of D-aspartate on kainate-induced AMPAR current was concentration-dependent and was voltage-independent in the tested voltage range (-80 to +60 mV). 3 The estimated EC50 of the L-glutamate-induced AMPAR current was increased in the presence of D-aspartate, while the estimated maximum L-glutamate-induced AMPAR current was not changed. D-aspartate concentration-dependently shifted the dose-response curve of kainate to the right. Schild plot analysis indicated that D-aspartate acts competitively to block AMPARs. The K(b) for D-aspartate was estimated to be 0.93 mM. 4 D-aspartate also blocked L-glutamate-induced current in Xenopus laevis oocytes that expressed recombinant homomeric AMPARs. 5 NMDA possessed similar inhibitory action on AMPARs. However, L-aspartate had little inhibitory action on AMPARs. 6 D-Aspartate, but not L-aspartate, was found to reduce the amplitude of miniature excitatory postsynaptic current in cultured hippocampal neurons. 7 Our data are consistent with a model in which D-aspartate directly competes with kainate and L-glutamate in binding to the agonist binding site of AMPARs. The prevalence of D-aspartate in the brain suggests a possible role of D-aspartate in modulating AMPAR-mediated fast excitatory synaptic transmission.
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Affiliation(s)
- Xiang-Qun Gong
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1
| | - Anne Frandsen
- Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
| | - Wei-Yang Lu
- Departments of Anaesthesia and Physiology, Sunnybrook and Women's College Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada M4N 3M5
| | - Yudi Wan
- Departments of Anaesthesia and Physiology, Sunnybrook and Women's College Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada M4N 3M5
| | - Rebecca L Zabek
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1
| | - Darryl S Pickering
- Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
| | - Donglin Bai
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1
- Author for correspondence:
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20
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Abstract
Certain excitatory pathways in the rat hippocampus can release aspartate along with glutamate. This study utilized rat hippocampal synaptosomes to characterize the mechanism of aspartate release and to compare it with glutamate release. Releases of aspartate and glutamate from the same tissue samples were quantitated simultaneously. Both amino acids were released by 25 mM K(+), 300 microM 4-aminopyridine (4-AP) and 0.5 and 1 microM ionomycin in a predominantly Ca(2+)-dependent manner. For a roughly equivalent quantity of glutamate released, aspartate release was significantly greater during exposure to elevated [K(+)] than to 4-AP and during exposure to 0.5 than to 1 microM ionomycin. Aspartate release was inefficiently coupled to P/Q-type voltage-dependent Ca(2+) channels and was reduced by KB-R7943, an inhibitor of reversed Na(+)/Ca(2+) exchange. In contrast, glutamate release depended primarily on Ca(2+) influx through P/Q-type channels and was not significantly affected by KB-R7943. Pretreatment of the synaptosomes with tetanus toxin and botulinum neurotoxins C and F reduced glutamate release, but not aspartate release. Aspartate release was also resistant to bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase, whereas glutamate release was markedly reduced. (+/-) -Threo-3-methylglutamate, a non-transportable competitive inhibitor of excitatory amino acid transport, did not reduce aspartate release. Niflumic acid, a blocker of Ca(2+)-dependent anion channels, did not alter the release of either amino acid. Exogenous aspartate and aspartate recently synthesized from glutamate accessed the releasable pool of aspartate as readily as exogenous glutamate and glutamate recently synthesized from aspartate accessed the releasable glutamate pool. These results are compatible with release of aspartate from either a vesicular pool by a "non-classical" form of exocytosis or directly from the cytoplasm by an as-yet-undescribed Ca(2+)-dependent mechanism. In either case, they suggest aspartate is released mainly outside the presynaptic active zones and may therefore serve as the predominant agonist for extrasynaptic N-methyl-D-aspartate receptors.
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Affiliation(s)
- S E Bradford
- Department of Pharmacology and Cancer Biology, Box 3813, 100B Research Park 2, Research Drive, Duke University Medical Center, Durham, NC 27710, USA
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21
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Chen W, Aoki C, Mahadomrongkul V, Gruber CE, Wang GJ, Blitzblau R, Irwin N, Rosenberg PA. Expression of a variant form of the glutamate transporter GLT1 in neuronal cultures and in neurons and astrocytes in the rat brain. J Neurosci 2002; 22:2142-52. [PMID: 11896154 PMCID: PMC2849837] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023] Open
Abstract
To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.
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Affiliation(s)
- Weizhi Chen
- Department of Neurology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA
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22
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Schachter D, Sang JC. Aortic leucine-to-glutamate pathway: metabolic route and regulation of contractile responses. Am J Physiol Heart Circ Physiol 2002; 282:H1135-48. [PMID: 11834513 DOI: 10.1152/ajpheart.00457.2001] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Rat aortic endothelium is differentiated regionally for three signal pathways capable of regulating the cGMP content of the underlying smooth muscle. Formation of nitric oxide (NO) from L-arginine and of glutamate from L-leucine increase cGMP; however, formation of prostaglandin H2 (PGH2) decreases cGMP. All three have peak activity in the windkessel area just distal to the aortic arch and decrease peripherally. We report evidence that the biochemical route of the leucine-to-glutamate (Leu-->Glu) pathway is via metabolism of leucine to acetyl CoA, that the controlling reaction of the pathway is mediated by the branched chain alpha-ketoacid dehydrogenase complex (BCDC), and that glutamate formation via the Leu-->Glu pathway is a major source of aortic segment free glutamate in vitro. Interruption of the pathway by treatment of precontracted rat aortic rings in vitro with each of three classes of inhibitors (leucine analogs, competitors for the BCDC reaction, or inhibitors of L-glutamate transport) enhances contractile responses. The enhancement requires an intact endothelium and is not owing to reductions in NO formation. The results support the hypothesis that the Leu-->Glu pathway functions in the regulation of aortic contractility and compliance.
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Affiliation(s)
- David Schachter
- Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
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23
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Larsson M, Persson S, Ottersen OP, Broman J. Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals. J Comp Neurol 2001; 430:147-59. [PMID: 11135252 DOI: 10.1002/1096-9861(20010205)430:2<147::aid-cne1021>3.0.co;2-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The role of L-aspartate as an excitatory neurotransmitter in primary afferent synapses in the spinal cord dorsal horn is disputed. To further investigate this issue, we examined the presence of aspartate-like immunoreactivity in primary afferent nerve terminals and other tissue components of the dorsal horn. We also examined the relationship between aspartate and glutamate immunogold labeling density and the density of synaptic vesicles in primary afferent terminals and presumed inhibitory terminals forming symmetric synapses. Weak aspartate immunosignals, similar to or lower than those displayed by presumed inhibitory terminals, were detected in both C-fiber primary afferent terminals in lamina II (dense sinusoid axon terminals, identified by morphological criteria) and in A-fiber primary afferent terminals in laminae III-IV (identified with anterograde transport of choleragenoid-horseradish peroxidase conjugate). The aspartate immunogold signal in primary afferent terminals was only about one-fourth of that in deep dorsal horn neuronal cell bodies. Further, whereas significant positive correlations were evident between synaptic vesicle density and glutamate immunogold labeling density in both A- and C-fiber primary afferent terminals, none of the examined terminal populations displayed a significant correlation between synaptic vesicle density and aspartate immunogold labeling density. Thus, our results indicate relatively low levels and a non-vesicular localization of aspartate in primary afferent terminals. It is therefore suggested that aspartate, rather than being a primary afferent neurotransmitter, serves a role in the intermediary metabolism in primary afferent terminals.
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Affiliation(s)
- M Larsson
- Department of Physiological Sciences, Lund University, S-223 62 Lund, Sweden
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24
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Gundersen V, Fonnum F, Ottersen OP, Storm-Mathisen J. Redistribution of neuroactive amino acids in hippocampus and striatum during hypoglycemia: a quantitative immunogold study. J Cereb Blood Flow Metab 2001; 21:41-51. [PMID: 11149667 DOI: 10.1097/00004647-200101000-00006] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Postembedding immunocytochemistry was used to localize aspartate, glutamate, gamma-aminobutyric acid (GABA), and glutamine in hippocampus and striatum during normo- and hypoglycemia in rat. In both brain regions, hypoglycemia caused aspartatelike immunoreactivity to increase. In hippocampus, this increase was evident particularly in the terminals of known excitatory afferents-in GABA-ergic neurons and myelinated axons. Aspartate was enriched along with glutamate in nerve terminals forming asymmetric synapses on spines and with GABA in terminals forming symmetric synapses on granule and pyramidal cell bodies. In both types of terminal, aspartate was associated with clusters of synaptic vesicles. Glutamate and glutamine immunolabeling were markedly reduced in all tissue elements in both brain regions, but less in the terminals than in the dendrosomatic compartments of excitatory neurons. In glial cells, glutamine labeling showed only slight attenuation. The level of GABA immunolabeling did not change significantly during hypoglycemia. The results support the view that glutamate and glutamine are used as energy substrates in hypoglycemia. Under these conditions both excitatory and inhibitory terminals are enriched with aspartate, which may be released from these nerve endings and thus contribute to the pattern of neuronal death characteristic of hypoglycemia.
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Affiliation(s)
- V Gundersen
- Anatomical Institute, University of Oslo, Norway
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25
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Martire M, Altobelli D, Maurizi S, Preziosi P, Fuxe K. K(+)-Evoked [(3)H]D-aspartate release in rat spinal cord synaptosomes: modulation by neuropeptide Y and calcium channel antagonists. J Neurosci Res 2000; 62:722-9. [PMID: 11104511 DOI: 10.1002/1097-4547(20001201)62:5<722::aid-jnr12>3.0.co;2-o] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
This study was conducted to investigate mechanisms regulating the release of [(3)H]D-aspartate (or endogenous glutamate) in the rat spinal cord. Presynaptic modulation of glutamate release was studied in superfused synaptosomes depolarized with 20 mM KCl. Calcium-channel antagonists, omega-conotoxin GVIA (omega-CgTx GVIA; N-type), nifedipine (L-type), and omega-conotoxin MVIIC (omega-CmTx MVIIC; P/Q type), were used to characterize the voltage-operated Ca(2+) channels (VOCCs) involved in this release. Nifedipine had no significant effect on the K(+)-evoked release of [(3)H]D-aspartate, but the omega-conotoxins GVIA and MVIIC produced dose-dependent inhibitory effects that were additive. The most substantial reduction (54.30% +/- 4.40%) was seen with omega-CgTx GVIA, indicating that N-type channels play a major role in the release of glutamate in this tissue. We investigated the effects of neuropeptide Y (NPY), NPY(13-36), and [Leu(31)][Pro(34)]NPY on Ca(2+)-dependent, K(+)-evoked [(3)H]D-aspartate release. NPY and NPY(13-36) equipotently inhibited the release of glutamate in a concentration-dependent manner. The half-maximal response was observed at about 12 nM; maximal inhibition of 44.22% +/- 4.60% was achieved with 0.3 microM. The selective GABA(B) agonist (-)baclofen inhibited K(+)-evoked [(3)H]D-aspartate release from superfused spinal cord synaptosomes by 50.00% +/- 4.80% at 10 microM. When NPY(13-36) and (-)baclofen were used together at maximal doses, their release-inhibiting effects were not additive. In addition, neither of the agonists was able to enhance the inhibition produced by pretreating the synaptosomes with the selective inhibitor of N-type VOCCs omega-CgTx GVIA. These results are consistent with the hypothesis that presynaptic Y(2)-like and GABA(B) receptors regulate glutamate release by blocking Ca(2+) currents through N-type VOCCs. Characterization of the receptors that can inhibit the release of glutamate may provide useful information for treatment of conditions characterized by excessive glutamatergic transmission in the spinal cord.
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Affiliation(s)
- M Martire
- Institute of Pharmacology, Catholic University of S. Heart, School of Medicine, Rome, Italy.
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26
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Abstract
Movement, the fundamental component of behavior and the principal extrinsic action of the brain, is produced when skeletal muscles contract and relax in response to patterns of action potentials generated by motoneurons. The processes that determine the firing behavior of motoneurons are therefore important in understanding the transformation of neural activity to motor behavior. Here, we review recent studies on the control of motoneuronal excitability, focusing on synaptic and cellular properties. We first present a background description of motoneurons: their development, anatomical organization, and membrane properties, both passive and active. We then describe the general anatomical organization of synaptic input to motoneurons, followed by a description of the major transmitter systems that affect motoneuronal excitability, including ligands, receptor distribution, pre- and postsynaptic actions, signal transduction, and functional role. Glutamate is the main excitatory, and GABA and glycine are the main inhibitory transmitters acting through ionotropic receptors. These amino acids signal the principal motor commands from peripheral, spinal, and supraspinal structures. Amines, such as serotonin and norepinephrine, and neuropeptides, as well as the glutamate and GABA acting at metabotropic receptors, modulate motoneuronal excitability through pre- and postsynaptic actions. Acting principally via second messenger systems, their actions converge on common effectors, e.g., leak K(+) current, cationic inward current, hyperpolarization-activated inward current, Ca(2+) channels, or presynaptic release processes. Together, these numerous inputs mediate and modify incoming motor commands, ultimately generating the coordinated firing patterns that underlie muscle contractions during motor behavior.
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Affiliation(s)
- J C Rekling
- Department of Neurobiology, University of California, Los Angeles, California 90095-1763, USA
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27
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Ventura R, Harris KM. Three-dimensional relationships between hippocampal synapses and astrocytes. J Neurosci 1999; 19:6897-906. [PMID: 10436047 PMCID: PMC6782870] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2023] Open
Abstract
Recent studies show that glutamate transporter-mediated currents occur in astrocytes when glutamate is released from hippocampal synapses. These transporters remove excess glutamate from the extracellular space, thereby facilitating synaptic input specificity and preventing neurotoxicity. Little is known about the position of astrocytic processes at hippocampal synapses. Serial electron microscopy and three-dimensional analyses were used to investigate structural relationships between astrocytes and synapses in stratum radiatum of hippocampal area CA1 in the mature rat in vivo and in slices. Only 57 +/- 11% of the synapses had astrocytic processes apposed to them. Of these, the astrocytic processes surrounded less than half (0.43 +/- 22) of the synaptic interface. Other studies suggest that astrocytes extend processes toward higher concentrations of glutamate; thus the presence of astrocytic processes at particular hippocampal synapses might signal which ones are releasing glutamate. The distance between nearest neighboring synapses was usually (approximately 95%) <1 microgram. Astrocytic processes occurred along the extracellular path between 33% of the neighboring synapses, neuronal processes occurred along the path between another 66% of the neighboring synapses, and only 1% of the synapses were close enough such that neither astrocytic nor neuronal processes occurred between them. These morphological arrangements suggest that the glutamate released at approximately two-thirds of hippocampal synapses might diffuse to other synapses, unless neuronal glutamate transporters are more effective than previously reported. The findings also suggest that physiological recordings made from hippocampal astrocytes do not uniformly sample the glutamate released from all hippocampal synapses.
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Affiliation(s)
- R Ventura
- Harvard College, Harvard Medical School, Division of Neuroscience in the Department of Neurology, Children's Hospital, Boston, Massachusetts 02115, USA
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28
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Verderio C, Bacci A, Coco S, Pravettoni E, Fumagalli G, Matteoli M. Astrocytes are required for the oscillatory activity in cultured hippocampal neurons. Eur J Neurosci 1999; 11:2793-800. [PMID: 10457176 DOI: 10.1046/j.1460-9568.1999.00697.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Synchronous oscillations of intracellular calcium concentration ([Ca2+]i) and of membrane potential occurred in a limited population of glutamatergic hippocampal neurons grown in primary cultures. The oscillatory activity occurred in synaptically connected cells only when they were in the presence of astrocytes. Microcultures containing only one or a few neurons also displayed oscillatory activity, provided that glial cells participated in the network. The glutamate-transporter inhibitors L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC) and dihydrokainate, which produce an accumulation of glutamate in the synaptic microenvironment, impaired the oscillatory activity. Moreover, in neurons not spontaneously oscillating, though in the presence of astrocytes, oscillations were induced by exogenous L-glutamate, but not by the stereoisomer D-glutamate, which is not taken up by glutamate transporters. These data demonstrate that astrocytes are essential for neuronal oscillatory activity and provide evidence that removal of glutamate from the synaptic environment is one of the major mechanisms by which glial cells allow the repetitive excitation of the postsynaptic cell.
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Affiliation(s)
- C Verderio
- CNR Cellular and Molecular Pharmacology and B. Ceccarelli Centers, Department of Medical Pharmacology, University of Milano, Italy
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29
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Chin KW, Lopez I, Lee SC, Honrubia V. Glutamate-like immunoreactivity during hair cell recovery after gentamicin exposure in the chinchilla vestibular sensory periphery. Laryngoscope 1999; 109:1037-44. [PMID: 10401837 DOI: 10.1097/00005537-199907000-00005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista ampullaris after gentamicin ototoxicity. STUDY DESIGN In five groups of three animals each, ototoxicity was produced by placing gentamicin (50 microg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1, 2, 4, 8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed. METHODS For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immunoreaction was detected using the avidinbiotinylated-complex technique and diaminobenzidine was the chromogen. RESULTS Normal sensory epithelia demonstrated type I and type II hair cells with moderate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-like immunoreactivity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type II hair cells surviving ototoxic treatment (15%-18%) contained moderate glutamate-like immunoreactivity, supporting cells showed no immunoreactivity, and nerve terminals and fibers displayed strong immunoreactivity. At 4 and 8 weeks after treatment, recovered hair cells (80%) had greater glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level. CONCLUSION Glutamate may be used as an indicator of hair cell differentiation and as an index of the molecular recovery of hair cells after ototoxicity.
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Affiliation(s)
- K W Chin
- Department of Surgery, University of California at Los Angeles, School of Medicine, USA
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Giuffrida R, Malatino LS, Bellomo M, Sapienza S. Immunohistochemical modifications of vasoactive neuropeptides and excitatory amino acids in the nervous tissue of the Mongolian gerbil after transient cerebral ischemia. Int J Dev Neurosci 1999; 17:99-107. [PMID: 10221669 DOI: 10.1016/s0736-5748(98)00089-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Modifications in the tissue concentration of vasoactive peptides (Endothelin, Calcitonin Gene Related Peptide, Atrial Natriuretic Peptide) and excitatory amino acids (glutamate, aspartate) were found in the nervous tissue of Mongolian gerbils after transient cerebral ischemia which was induced by unilateral occlusion of the common carotid artery for 30 min 4 h. In fact, immunostaining for these peptides was more intense in the ischemic tissue: the greatest increases of tissue immunoreactivity were observed for Endothelin; smaller differences were found for Calcitonin Gene Related Peptide and Atrial Natriuretic Peptide. Immunostaining for Neuropeptide Y, another vasoactive neuropeptide, was virtually unchanged. Infarct areas, when present, contained numerous Endothelin-immunoreactive cell bodies. On the contrary, the same areas were completely void of glutamate- or aspartate-immunostained neurons, normally present in the correspondent regions of the control tissue. The present results suggest that severe cerebral ischemia is paralleled by an unbalance of local vasoactive factors. The predominance of vasoconstrictor action of Endothelin might play a major role in the irreversible damage, together with the excitotoxic effect of the extracellular accumulation of excitatory amino acids, probably due to a leakage from neuronal cell somata, as suggested by the disappearance of glutamate- or aspartate-immunostained neurons.
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Affiliation(s)
- R Giuffrida
- Department of Physiological Sciences, Catania, Italy
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31
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Bacci A, Verderio C, Pravettoni E, Matteoli M. The role of glial cells in synaptic function. Philos Trans R Soc Lond B Biol Sci 1999; 354:403-9. [PMID: 10212490 PMCID: PMC1692488 DOI: 10.1098/rstb.1999.0393] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Glial cells represent the most abundant cell population in the central nervous system and for years they have been thought to provide just structural and trophic support to neurons. Recently, several studies were performed, leading to the identification of an active interaction between glia and neurons. This paper focuses on the role played by glial cells at the level of the synapse, reviewing recent data defining how glia is determinant in synaptogenesis, in the modulation of fully working synaptic contacts and in synaptic plasticity.
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Affiliation(s)
- A Bacci
- CNR-Cellular and Molecular Pharmacology, University of Milano, Italy
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32
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Danbolt NC, Chaudhry FA, Dehnes Y, Lehre KP, Levy LM, Ullensvang K, Storm-Mathisen J. Properties and localization of glutamate transporters. PROGRESS IN BRAIN RESEARCH 1999; 116:23-43. [PMID: 9932368 DOI: 10.1016/s0079-6123(08)60428-8] [Citation(s) in RCA: 74] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- N C Danbolt
- Department of Anatomy, University of Oslo, Norway.
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33
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Abstract
The acidic amino acids, glutamate and aspartate, are the predominant excitatory neurotransmitters in the mammalian CNS. Under many pathologic conditions, these excitatory amino acids (EAAs) accumulate in the extracellular fluid in CNS and the resultant excessive activation of EAA receptors contributes to brain injury through a process known as 'excitotoxicity'. Unlike many other neurotransmitters, there is no evidence for extracellular metabolism of EAAs, rather, they are cleared by Na+-dependent transport mechanisms. Therefore, this transport process is important for ensuring crisp synaptic signaling as well as limiting the excitotoxic potential of EAAs. With the cloning of five distinct EAA transporters, a variety of tools were developed to characterize individual transporter subtypes, including specific antibodies, expression systems, and probes to delete/knock-down expression of each subtype. These tools are beginning to provide fundamental information that has the potential to impact our understanding of EAA physiology and pathophysiology. For example, biophysical studies of the cloned transporters have led to the observation that some subtypes function as ligand-gated ion channels as well as transporters. With these reagents, it has also been possible to explore the relative contributions of each transporter to the clearance of extracellular EAAs and to begin to examine the regulation of specific transporter subtypes. In this review, an overview of the properties of the transporter subtypes will be presented. The evidence which suggests that the transporter, GLT1/EAAT2, may be sufficient to explain a large percentage of forebrain transport will be critically reviewed. Finally, the studies of regulation of GLT-1 in vitro and in vivo will be described.
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Affiliation(s)
- M B Robinson
- Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania 19104-4318, USA.
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34
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Gundersen V, Chaudhry FA, Bjaalie JG, Fonnum F, Ottersen OP, Storm-Mathisen J. Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus. J Neurosci 1998; 18:6059-70. [PMID: 9698301 PMCID: PMC6793189] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/1998] [Revised: 05/18/1998] [Accepted: 05/26/1998] [Indexed: 02/08/2023] Open
Abstract
To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.
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Affiliation(s)
- V Gundersen
- Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Blindern, N-0317 Oslo, Norway
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35
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Masliah E, Raber J, Alford M, Mallory M, Mattson MP, Yang D, Wong D, Mucke L. Amyloid protein precursor stimulates excitatory amino acid transport. Implications for roles in neuroprotection and pathogenesis. J Biol Chem 1998; 273:12548-54. [PMID: 9575214 DOI: 10.1074/jbc.273.20.12548] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Excitatory neurotransmitters such as glutamate are required for the normal functioning of the central nervous system but can trigger excitotoxic neuronal injury if allowed to accumulate to abnormally high levels. Their extracellular levels are controlled primarily by transmitter uptake into astrocytes. Here, we demonstrate that the amyloid protein precursor may participate in the regulation of this important process. The amyloid protein precursor has been well conserved through evolution, and a number of studies indicate that it may function as an endogenous excitoprotectant. However, the mechanisms underlying this neuroprotective capacity remain largely unknown. At moderate levels of expression, human amyloid protein precursors increased glutamate/aspartate uptake in brains of transgenic mice, with the 751-amino acid isoform showing greater potency than the 695-amino acid isoform. Cerebral glutamate/aspartate transporter protein levels were higher in transgenic mice than in non-transgenic controls, whereas transporter mRNA levels were unchanged. Amyloid protein precursor-dependent stimulation of aspartate uptake by cultured primary astrocytes was associated with increases in protein kinase A and C activity and could be blocked by inhibitors of these kinases. The stimulation of astroglial excitatory amino acid transport by amyloid protein precursors could protect the brain against excitotoxicity and may play an important role in neurotransmission.
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Affiliation(s)
- E Masliah
- Departments of Neurosciences and Pathology, University of California San Diego, La Jolla, California 92093-0624, USA
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36
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Weaver CD, Gundersen V, Verdoorn TA. A high affinity glutamate/aspartate transport system in pancreatic islets of Langerhans modulates glucose-stimulated insulin secretion. J Biol Chem 1998; 273:1647-53. [PMID: 9430708 DOI: 10.1074/jbc.273.3.1647] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3H]glutamate uptake activity was Na(+)-dependent, and it was blocked by L-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a Vmax of 8.48 +/- 1.47 fmol/min/islet (n = 4), which corresponds to 102.2 +/- 17.7 pmol/min/mg islet protein. The apparent Km of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mM), the apparent Km was 34.7 +/- 7.8 microM (n = 3). However, in high glucose (16.7 mM) the apparent Km increased to 112.7 +/- 16.5 microM (n = 3) with little or no change in Vmax. Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported D-aspartate. Anti-D-aspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-beta cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids, L-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mM glucose potentiated insulin secretion 23.3 +/- 2.3% (n = 3) compared with 8.3 mM glucose alone. This effect was abolished in the presence of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 +/- 5.9% (n = 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion.
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Affiliation(s)
- C D Weaver
- CNS Drug Discovery, Bristol-Meyers Squibb, Wallingford, Connecticut 06492, USA
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37
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Schaffar N, Rao H, Kessler JP, Jean A. Immunohistochemical detection of glutamate in rat vagal sensory neurons. Brain Res 1997; 778:302-8. [PMID: 9459547 DOI: 10.1016/s0006-8993(97)01058-5] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.
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Affiliation(s)
- N Schaffar
- Département de Physiologie et Neurophysiologie, CNRS ESA 6034- Case 351, Faculté Saint-Jérôme, Marseille, France
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38
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Muzzolini A, Bregola G, Bianchi C, Beani L, Simonato M. Characterization of glutamate and [3H]D-aspartate outflow from various in vitro preparations of the rat hippocampus. Neurochem Int 1997; 31:113-24. [PMID: 9185171 DOI: 10.1016/s0197-0186(96)00129-5] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The characteristics of high-K+ and electrically evoked endogenous glutamate and [3H]D-aspartate release have been studied in multiple in vitro preparations of the rat hippocampus (transverse slices, granule cells cultures, synaptosomes and mossy fibre synaptosomes) under similar experimental conditions. High external K+ concentrations evoked [3H]D-aspartate and endogenous glutamate overflow in a concentration-dependent manner in all preparations (except it was not possible to measure endogenous glutamate outflow from granule cells). This effect was tetrodotoxin-insensitive but partially calcium-dependent. In slices, field electrical stimulation evoked an overflow of endogenous glutamate, but not of [3H]D-aspartate, in a frequency-dependent manner. This effect was concentration-dependently amplified by the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC). The electrically evoked glutamate overflow in the presence of t-PDC was tetrodotoxin-sensitive and calcium-dependent. In primary dentate gyrus cell cultures, electrical stimulation evoked an overflow of [3H]D-aspartate in a frequency-dependent manner, while endogenous glutamate outflow was not detectable. This effect could be inhibited by tetrodotoxin and by the N-type calcium channel blocker omega-conotoxin GVIA. Finally, the effect of adenosine has been studied in order to assess the pharmacological modulability of [3H]D-aspartate and endogenous glutamate stimulation-induced overflow. Adenosine was found to inhibit 35 mM K(+)- and 20 Hz electrical stimulation-induced [3H]D-aspartate and endogenous glutamate overflow. These effects were all prevented by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). These data are in line with the hypothesis that reuptake plays a role in regulating glutamate release, and that [3H]D-aspartate represents a valid marker of endogenous glutamate under most (but not all) experimental conditions.
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Affiliation(s)
- A Muzzolini
- Institute of Pharmacology, University of Ferrara, Italy
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39
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Shupliakov O, Löw P, Grabs D, Gad H, Chen H, David C, Takei K, De Camilli P, Brodin L. Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions. Science 1997; 276:259-63. [PMID: 9092476 DOI: 10.1126/science.276.5310.259] [Citation(s) in RCA: 383] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain-containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin's SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.
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Affiliation(s)
- O Shupliakov
- The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden
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40
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Shupliakov O, Ottersen OP, Storm-Mathisen J, Brodin L. Glial and neuronal glutamine pools at glutamatergic synapses with distinct properties. Neuroscience 1997; 77:1201-12. [PMID: 9130798 DOI: 10.1016/s0306-4522(96)00537-4] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The main pathway for transmitter glutamate turnover in excitatory synapses is thought to involve an uptake in glial processes, a conversion into glutamine, which recycles to the presynaptic terminal to serve as the main precursor for new synthesis of glutamate. To investigate whether the mechanisms of glutamine and glutamate turnover are linked with the properties of different glutamate synapses, the distribution of glutamine was studied in two types of glutamate synapse in the lamprey spinal cord using immunogold post-embedding electron microscopy. The synapses examined are formed by primary afferent axons (dorsal column axons), which predominantly exhibit a tonic firing pattern, and by giant reticulospinal axons, which primarily fire in brief bursts. Glial cell processes and postsynaptic dendrites displayed the highest density of glutamine labeling in both types of synapse. The level of glutamine was significantly higher in the glial cell processes surrounding the tonic dorsal column synapses, as compared to those surrounding the reticulospinal synapses. The axoplasmic matrix and presynaptic mitochondria, as well as postsynaptic dendrites, contained similar levels of glutamine labeling in both cases. The glutamate labeling in glial processes was also similar at the two types of synapse, while axoplasmic matrix and presynaptic mitochondria displayed four to six times higher levels in the tonic axons. In conjunction with our previous results, showing a different transport activity in glial processes of the two types of excitatory synapse, the results of the present study suggest that the glial pool of neurotransmitter precursor is linked to the rate of transmitter synthesis and release in adjacent synapses.
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Affiliation(s)
- O Shupliakov
- Department of Neuroscience, Karolinska Institute, Stockholm, Sweden
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41
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Obrenovitch TP, Urenjak J. Altered glutamatergic transmission in neurological disorders: from high extracellular glutamate to excessive synaptic efficacy. Prog Neurobiol 1997; 51:39-87. [PMID: 9044428 DOI: 10.1016/s0301-0082(96)00049-4] [Citation(s) in RCA: 240] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
This review is a critical appraisal of the widespread assumption that high extracellular glutamate, resulting from enhanced pre-synaptic release superimposed on deficient uptake and/or cytosolic efflux, is the key to excessive glutamate-mediated excitation in neurological disorders. Indeed, high extracellular glutamate levels do not consistently correlate with, nor necessarily produce, neuronal dysfunction and death in vivo. Furthermore, we exemplify with spreading depression that the sensitivity of an experimental or pathological event to glutamate receptor antagonists does not imply involvement of high extracellular glutamate levels in the genesis of this event. We propose an extension to the current, oversimplified concept of excitotoxicity associated with neurological disorders, to include alternative abnormalities of glutamatergic transmission which may contribute to the pathology, and lead to excitotoxic injury. These may include the following: (i) increased density of glutamate receptors; (ii) altered ionic selectivity of ionotropic glutamate receptors; (iii) abnormalities in their sensitivity and modulation; (iv) enhancement of glutamate-mediated synaptic efficacy (i.e. a pathological form of long-term potentiation); (v) phenomena such as spreading depression which require activation of glutamate receptors and can be detrimental to the survival of neurons. Such an extension would take into account the diversity of glutamate-receptor-mediated processes, match the complexity of neurological disorders pathogenesis and pathophysiology, and ultimately provide a more elaborate scientific basis for the development of innovative treatments.
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Affiliation(s)
- T P Obrenovitch
- Department of Neurochemistry, Institute of Neurology, London.
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42
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Abstract
Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg(2+)-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, beta-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that "quanta" in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events.
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Affiliation(s)
- E M Fykse
- Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller, Norway.
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Mize RR, Butler GD. Postembedding immunocytochemistry demonstrates directly that both retinal and cortical terminals in the cat superior colliculus are glutamate immunoreactive. J Comp Neurol 1996; 371:633-48. [PMID: 8841915 DOI: 10.1002/(sici)1096-9861(19960805)371:4<633::aid-cne11>3.0.co;2-k] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Although the excitatory neurotransmitter glutamate is known to be present in the cat superior colliculus (SC), the types of synapses that contain glutamate have not been examined. We, therefore, studied the ultrastructure of synaptic profiles labeled by a glutamate antibody by using electron microscopic postembedding immunocytochemistry. In addition, unilateral aspiration lesions of areas 17-18 were made at 5-28 days before death in order to determine whether degenerating terminals from visual cortex were glutamate immunoreactive (Glu-ir). Three types of axon terminal were glu-ir: 1) those containing large, round synaptic vesicles and pale mitochondria, characteristic of retinal terminals (RT profiles); 2) those containing small, round synaptic vesicles and dark mitochondria (RSD profiles); and 3) those containing large, round synaptic vesicles and dark mitochondria (RLD profiles). Measures of mean gold particle density revealed that RT, RSD, and RLD profiles had similar average grain densities (11.3-12.7 particles/unit area). Other labeled profile types included cell bodies, large-calibre dendrites, and myelinated axons. Axon terminals containing flattened synaptic vesicles and vesicle-containing presynaptic dendrites, both of which contain gamma-aminobutyric acid (GABA), had many fewer gold particles (3.6 and 4.8 mean particles/unit area, respectively). Following unilateral removal of visual cortex, normal RSD terminals were observed infrequently in the SC ipsilateral to the lesion. Synaptic terminals in the initial stages of degeneration were heavily labeled by the glutamate antibody, as were axon terminals and myelinated axons undergoing hypertrophied or neurofilamentous degeneration. These results show that both major sensory afferents to the superficial layers of cat SC contain glutamate--RT terminals from the retina and RSD terminals from visual cortex. The origin of RLD terminals is unknown.
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Affiliation(s)
- R R Mize
- Department of Anatomy, Louisiana State University Medical Center, New Orleans 70112, USA
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44
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Gundersen V, Ottersen OP, Storm-Mathisen J. Selective excitatory amino acid uptake in glutamatergic nerve terminals and in glia in the rat striatum: quantitative electron microscopic immunocytochemistry of exogenous (D)-aspartate and endogenous glutamate and GABA. Eur J Neurosci 1996; 8:758-65. [PMID: 9081627 DOI: 10.1111/j.1460-9568.1996.tb01261.x] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
To characterize glutamate/aspartate uptake activity in various cellular and subcellular elements in the striatum, rat striatal slices were exposed to 10 and 50 mu M exogenous (D)-aspartate. After fixation with glutaraldehyde/formaldehyde the distribution of (D)-aspartate was analysed by postembedding immunocytochemistry and the ultrastructural distribution was compared with the distributions of endogenous glutamate and GABA. Light microscopically, (D)-aspartate-like immunoreactivity was localized in conspicuous dots along very weakly labelled dendritic profiles and neuron cell bodies. At the electron microscope level gold particles signalling (D)-aspartate occurred at highest density in nerve terminals making asymmetrical contacts with postsynaptic spines (i.e. resembling synapses of cortical afferents). Astrocytic processes also contained gold particles, but at a lower density than nerve endings. In contrast, dendritic spines were only weakly (D)-aspartate-positive. The difference in labelling at 10 and 50 mu M (D)-aspartate was consistent with 'high-affinity' uptake. Neighbouring sections processed with other antibodies showed that the D-aspartate labelling. Occurred in nerve terminals strongly immunoreactive for glutamate, rather than in terminals very weakly glutamate-immunopositive or in nerve endings immunoreactive for GABA. Glutamate labelling of perfusion-fixed striatum confirmed that terminals forming asymmetrical synaptic contacts with spines were enriched with gold particles, suggesting that these terminals use glutamate as a transmitter. This study demonstrates that high-affinity uptake sites for excitatory amino acids in the striatum are most strongly expressed on presumed glutamatergic nerve terminals and on astrocytes.
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Affiliation(s)
- V Gundersen
- Anatomical Institute, University of Oslo, Norway
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