Expression of the vesicular glutamate transporters-1 and -2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury

The expression of two vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT1 and VGLUT2, respectively. VGLUT1-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT2-IR NPs. Most VGLUT1-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, approximately 31% and approximately 42% of the VGLUT2-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT2. Moderate colocalization between VGLUT1 and VGLUT2 was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT1- and VGLUT2-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT2-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the dorsal horn of the spinal cord, strong VGLUT1-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT2-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT1-LI, while apparently leaving unaffected the VGLUT2-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT2-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT1-IR fibers were also detected in the skin. Some VGLUT1-IR and VGLUT2-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate. J Comp Neurol 483:1-16], we speculate that virtually all DRG neurons in adult mouse express VGLUTs and use glutamate as transmitter.

[1]  R. Coggeshall,et al.  Glutamate immunoreactivity in rat dorsal root axons , 1989, Neuroscience Letters.

[2]  R. Coggeshall,et al.  Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery , 1998, The Journal of comparative neurology.

[3]  T Hori,et al.  Molecular Cloning of a Novel Brain‐Type Na+‐Dependent Inorganic Phosphate Cotransporter , 2000, Journal of neurochemistry.

[4]  Takeshi Kaneko,et al.  Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia , 2003, Synapse.

[5]  W. Streit,et al.  Differential Regulation of Calcitonin Gene‐related Peptide (CGRP) in Regenerating Rat Facial Nucleus and Dorsal Root Ganglion , 1991, The European journal of neuroscience.

[6]  Y. Jan,et al.  Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Peter König,et al.  Most peptide‐containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate , 2005, The Journal of comparative neurology.

[8]  T. Jessell,et al.  Amino acid‐mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord. , 1990, The Journal of physiology.

[9]  D. Copenhagen,et al.  Vesicular Glutamate Transporters 1 and 2 Target to Functionally Distinct Synaptic Release Sites , 2004, Science.

[10]  F. Fujiyama,et al.  Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain , 2002, The Journal of comparative neurology.

[11]  U. Nielsch,et al.  Effect of cutting or crushing the rat sciatic nerve on synthesis of substance P by isolated L5 dorsal root ganglia , 1987, Neuropeptides.

[12]  Makoto Sato,et al.  α-CGRP and β-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion , 1990 .

[13]  S. Carlton.  Peripheral excitatory amino acids. , 2001, Current opinion in pharmacology.

[14]  J. Keast,et al.  Glutamate and aspartate immunoreactivity in dorsal root ganglion cells supplying visceral and somatic targets and evidence for peripheral axonal transport , 2000, The Journal of comparative neurology.

[15]  J. Storm-Mathisen,et al.  Different neuronal localization of aspartate-like and glutamate-like immunoreactivities in the hippocampus of rat, guinea-pig and senegalese baboon (Papio papio), with a note on the distribution of γ-aminobutyrate , 1985, Neuroscience.

[16]  S. Schuldiner,et al.  Mechanism of Transport and Storage of Neurotransmitter , 1987 .

[17]  T. Kaneko,et al.  Glutaminase immunoreactive neurons in the rat dorsal root ganglion contain calcitonin gene-related peptide (CGRP) , 1993, Neuroscience Letters.

[18]  Christian Rosenmund,et al.  Vesicular Glutamate Transporter VGLUT2 Expression Levels Control Quantal Size and Neuropathic Pain , 2006, The Journal of Neuroscience.

[19]  P. Vidal,et al.  Modulation of the glutamatergic receptors (AMPA and NMDA) and of glutamate vesicular transporter 2 in the rat facial nucleus after axotomy , 2005, Neuroscience.

[20]  Yuan Guo,et al.  Local application of morphine suppresses glutamate-evoked activities of C and Aδ afferent fibers in rat hairy skin , 2005, Brain Research.

[21]  A. Kirchgessner,et al.  Vesicular glutamate transporter 2 in the brain–gut axis , 2001, Neuroreport.

[22]  J. Storm-Mathisen,et al.  The Expression of Vesicular Glutamate Transporters Defines Two Classes of Excitatory Synapse , 2001, Neuron.

[23]  F. Fonnum.  Glutamate: A Neurotransmitter in Mammalian Brain , 1984, Journal of neurochemistry.

[24]  R. Villalba,et al.  Vesicular glutamate transporters in the spinal cord, with special reference to sensory primary afferent synapses , 2004, The Journal of comparative neurology.

[25]  T. Hökfelt,et al.  Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy , 1987, Neuroscience Letters.

[26]  R. Petralia,et al.  Light and electron microscopic immunocytochemical localization of AMPA‐selective glutamate receptors in the rat spinal cord , 1994, The Journal of comparative neurology.

[27]  Ichiro Kanazawa,et al.  Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons , 1979, Brain Research.

[28]  Y. Moriyama,et al.  Expression and Localization of Vesicular Glutamate Transporters in Pancreatic Islets, Upper Gastrointestinal Tract, and Testis , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  G. Battaglia,et al.  Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey , 1988, The Journal of comparative neurology.

[30]  M. Ludwig.  Dendritic neurotransmitter release , 2005 .

[31]  E. Mugnaini,et al.  Merkel cells, corpuscular nerve endings and free nerve endings in the mouse palatine mucosa express three subtypes of vesicular glutamate transporters , 2004, Journal of neurocytology.

[32]  O. Ottersen,et al.  Enrichment of Glutamate‐like Immunoreactivity in Primary Afferent Terminals Throughout the Spinal Cord Dorsal Horn , 1993, The European journal of neuroscience.

[33]  J. Kessler,et al.  Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract , 2006, Neuroscience.

[34]  D. Wynick,et al.  Use of genetically engineered transgenic mice to investigate the role of galanin in the peripheral nervous system after injury , 2005, Neuropeptides.

[35]  W. Neuhuber,et al.  Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function? , 2003, Cell and Tissue Research.

[36]  Alyson Fox,et al.  Metabotropic Glutamate Receptor 5 Upregulation in A-Fibers after Spinal Nerve Injury: 2-Methyl-6-(Phenylethynyl)-Pyridine (MPEP) Reverses the Induced Thermal Hyperalgesia , 2002, The Journal of Neuroscience.

[37]  Coons Ah.  Fluorescent antibody methods , 1968 .

[38]  R. H. Evans,et al.  The primary afferent depolarizing action of kainate in the rat , 1986, British journal of pharmacology.

[39]  B. Giros,et al.  A Third Vesicular Glutamate Transporter Expressed by Cholinergic and Serotoninergic Neurons , 2002, The Journal of Neuroscience.

[40]  S. Fleetwood-Walker,et al.  Nociception in vertebrates: key receptors participating in spinal mechanisms of chronic pain in animals , 2004, Brain Research Reviews.

[41]  Masahiko Watanabe,et al.  Subtype switching of vesicular glutamate transporters at parallel fibre–Purkinje cell synapses in developing mouse cerebellum , 2003, The European journal of neuroscience.

[42]  S. Paul,et al.  Cloning and expression of a cDNA encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  J. Collins,et al.  Molecular and Functional Analysis of a Novel Neuronal Vesicular Glutamate Transporter* , 2001, The Journal of Biological Chemistry.

[44]  C. D. Stern,et al.  Handbook of Chemical Neuroanatomy Methods in Chemical Neuroanatomy. Edited by A. Bjorklund and T. Hokfelt. Elsevier, Amsterdam, 1983. Cloth bound, 548 pp. UK £140. (Volume 1 in the series). , 1986, Neurochemistry International.

[45]  Xiao-jun Xu,et al.  Plasticity in spinal nociception after peripheral nerve section: reduced effectiveness of the NMDA receptor antagonist MK-801 in blocking wind-up and central sensitization of the flexor reflex , 1995, Brain Research.

[46]  Xiao-jun Xu,et al.  Neuropeptides in neuropathic and inflammatory pain with special emphasis on cholecystokinin and galanin. , 2001, European journal of pharmacology.

[47]  M. Devor,et al.  Functional cross-excitation between afferent A- and C-neurons in dorsal root ganglia , 1999, Neuroscience.

[48]  Lan Bao,et al.  Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[49]  A. Dickenson.  NMDA receptor antagonists: interactions with opioids , 1997, Acta anaesthesiologica Scandinavica.

[50]  J. Storm-Mathisen,et al.  First visualization of glutamate and GABA in neurones by immunocytochemistry , 1983, Nature.

[51]  M. Heinrich.  Sensory mechanisms of the spinal cord, ed 2. By William D. Willis, Jr, and Richard E. Coggeshall New York, Plenum Publishing, I991 575 pp, illustrated, $89.50 , 1992 .

[52]  J. Watkins.  l-glutamate as a central neurotransmitter: looking back. , 2000, Biochemical Society transactions.

[53]  D. Adriaensen,et al.  Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies , 2003, Histochemistry and Cell Biology.

[54]  E. T. O. Rugarn.  Radioimmunoassay for rat galanin: immunochemical and chromatographic characterization of immunoreactivity in tissue extracts , 2000, Scandinavian journal of clinical and laboratory investigation.

[55]  J. Storm-Mathisen,et al.  Glutamate‐ and GABA‐containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique , 1984, The Journal of comparative neurology.

[56]  T. Hökfelt,et al.  NPY Y1 Receptors Are Present in Axonal Processes of DRG Neurons , 2002, Experimental Neurology.

[57]  R. Weinberg,et al.  Amino acid immunocytochemistry of primary afferent terminals in the rat dorsal horn , 1994, The Journal of comparative neurology.

[58]  R. Coggeshall,et al.  Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin , 1995, Neuroscience Letters.

[59]  T. Hökfelt,et al.  The participation of galanin in pain processing at the spinal level. , 2002, Trends in pharmacological sciences.

[60]  A. Todd,et al.  Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord , 2004, Brain Research.

[61]  David J. Anderson,et al.  Topographically Distinct Epidermal Nociceptive Circuits Revealed by Axonal Tracers Targeted to Mrgprd , 2005, Neuron.

[62]  P. Genever,et al.  Essential components for a glutamatergic synapse between Merkel cell and nerve terminal in rats , 2004, Neuroscience Letters.

[63]  R. Fremeau,et al.  Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. , 2000, Science.

[64]  V. Parpura,et al.  Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes , 2004, Journal of Neuroscience.

[65]  T. Kaneko,et al.  Immunohistochemical demonstration of glutaminase in catecholaminergic and serotoninergic neurons of rat brain , 1990, Brain Research.

[66]  O. Ottersen.  Postembedding light- and electron microscopic immunocytochemistry of amino acids: description of a new model system allowing identical conditions for specificity testing and tissue processing , 2004, Experimental Brain Research.

[67]  R. Gutiérrez,et al.  Programmed and Induced Phenotype of the Hippocampal Granule Cells , 2005, The Journal of Neuroscience.

[68]  F. Fujiyama,et al.  Vesicular glutamate transporters, VGluT1 and VGluT2, in the trigeminal ganglion neurons of the rat, with special reference to coexpression , 2003, The Journal of comparative neurology.

[69]  V. Neugebauer,et al.  Peripheral metabotropic glutamate receptors as drug targets for pain relief , 2002, Expert opinion on therapeutic targets.

[70]  J. Arvidsson,et al.  Ultrastructural changes of the central scalloped (C1) primary afferent endings of synaptic glomeruli in the substantia gelatinosa Rolandi of the rat after peripheral neurotomy , 1990, Journal of neurocytology.

[71]  B. Robertson,et al.  Anterograde transport of horseradish-peroxidase conjugated isolectin B4 from Griffonia simplicifolia I in spinal primary sensory neurons of the rat , 1998, Brain Research.

[72]  M. Schäfer,et al.  Identification of the Differentiation-Associated Na+/PI Transporter as a Novel Vesicular Glutamate Transporter Expressed in a Distinct Set of Glutamatergic Synapses , 2002, The Journal of Neuroscience.

[73]  M. A. Kai-Kai,et al.  Glutamate-immunoreactivity in the trigeminal and dorsal root ganglia, and intraspinal neurons and fibres in the dorsal horn of the rat , 1991, The Histochemical Journal.

[74]  G. Kerchner,et al.  Glutamate and the Presynaptic Control of Spinal Sensory Transmission , 2002, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[75]  K. Hackshaw,et al.  Glutamate uptake is attenuated in spinal deep dorsal and ventral horn in the rat spinal nerve ligation model , 2005, Brain Research.

[76]  F. Fujiyama,et al.  Expression of vesicular glutamate transporters, VGluT1 and VGluT2, in axon terminals of nociceptive primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns of the rat , 2003, The Journal of comparative neurology.

[77]  Y. Moriyama,et al.  Differentiation-associated Na+-dependent Inorganic Phosphate Cotransporter (DNPI) Is a Vesicular Glutamate Transporter in Endocrine Glutamatergic Systems* , 2001, The Journal of Biological Chemistry.

[78]  J. Storm-Mathisen,et al.  Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters , 2004, The Journal of comparative neurology.

[79]  M. Devor.  Response of nerves to injury in relation to neuropathic pain , 2006 .

[80]  W. Willis,et al.  Sensory Mechanisms of the Spinal Cord , 1991, Springer US.

[81]  S. Eichmüller,et al.  Hair cycle‐dependent plasticity of skin and hair follicle innervation in normal murine skin , 1997, The Journal of comparative neurology.

[82]  R. Fremeau,et al.  VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate , 2004, Trends in Neurosciences.

[83]  J. Polak,et al.  The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. , 1988, British journal of experimental pathology.

[84]  S. Nakanishi,et al.  Presynaptic localization of a metabotropic glutamate receptor, mGluR7, in the primary afferent neurons: an immunohistochemical study in the rat , 1995, Neuroscience Letters.

[85]  T. Hökfelt,et al.  Chemical anatomy of the brain. , 1984, Science.

[86]  Y. Moriyama,et al.  Glutamatergic chemical transmission: look! Here, there, and anywhere. , 2004, Journal of biochemistry.

[87]  S. Biasi,et al.  Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[88]  T. Hökfelt,et al.  Phenotyping of sensory and sympathetic ganglion neurons of a galanin-overexpressing mouse—Possible implications for pain processing , 2006, Journal of Chemical Neuroanatomy.

[89]  M. Schäfer,et al.  Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons* , 2002, The Journal of Biological Chemistry.

[90]  D. Copenhagen,et al.  The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[91]  B. Giros,et al.  The Existence of a Second Vesicular Glutamate Transporter Specifies Subpopulations of Glutamatergic Neurons , 2001, The Journal of Neuroscience.

[92]  G. Paxinos.  The Rat nervous system , 1985 .

[93]  F. Fujiyama,et al.  Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents , 2004, Brain Research.

[94]  Takeshi Kaneko,et al.  Complementary distribution of vesicular glutamate transporters in the central nervous system , 2002, Neuroscience Research.

[95]  A. Burette,et al.  Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors , 2004, Journal of neurocytology.

[96]  V. Pickel,et al.  The Localization of the Brain-Specific Inorganic Phosphate Transporter Suggests a Specific Presynaptic Role in Glutamatergic Transmission , 1998, The Journal of Neuroscience.

[97]  T. Hökfelt,et al.  CGRP-like immunoreactivity in A11 dopamine neurons projecting to the spinal cord and a note on CGRP-CCK cross-reactivity , 1993, Brain Research.

[98]  P. Wall,et al.  Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury , 2000, Pain.

[99]  Joe C. Adams,et al.  Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. , 1992, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[100]  M. Tohyama,et al.  AMPA, KA and NMDA receptors are expressed in the rat DRG neurones. , 1993, Neuroreport.

[101]  D. Maxwell,et al.  Axon terminals possessing the α2c-adrenergic receptor in the rat dorsal horn are predominantly excitatory , 2003, Brain Research.

[102]  S. Carlton,et al.  Metabotropic glutamate 1α receptors on peripheral primary afferent fibers: their role in nociception , 2001, Brain Research.

[103]  G. Volsi,et al.  Glutamate immunocytochemistry in the dorsal horn after injury or stimulation of the sciatic nerve of rats , 1993, Brain Research Bulletin.

[104]  Amin Derouiche,et al.  Astrocytic exocytosis vesicles and glutamate: A high‐resolution immunofluorescence study , 2005, Glia.

[105]  A. Dhillon,et al.  PGP 9.5—a new marker for vertebrate neurons and neuroendocrine cells , 1983, Brain Research.

[106]  R. H. Evans,et al.  A comparison of excitatory amino acid antagonists acting at primary afferent C fibres and motoneurones of the isolated spinal cord of the rat , 1987, British journal of pharmacology.

[107]  T. Kamada,et al.  Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate , 2004, Experimental Brain Research.

[108]  G. G. Nagy,et al.  The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn , 2003, The European journal of neuroscience.

[109]  Michael Costigan,et al.  Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. , 2002, BMC Neuroscience.

[110]  T. White,et al.  Intraplantar injection of glutamate evokes peripheral adenosine release in the rat hind paw: involvement of peripheral ionotropic glutamate receptors and capsaicin‐sensitive sensory afferents , 2002, Journal of neurochemistry.

[111]  Christian Rosenmund,et al.  Identification of Differentiation-Associated Brain-Specific Phosphate Transporter as a Second Vesicular Glutamate Transporter (VGLUT2) , 2001, The Journal of Neuroscience.

[112]  Masahiko Watanabe,et al.  Expression and distribution of JNK/SAPK‐associated scaffold protein JSAP1 in developing and adult mouse brain , 2006, Journal of neurochemistry.

[113]  J. D. Richardson,et al.  Glutamate participates in the peripheral modulation of thermal hyperalgesia in rats. , 1995, European journal of pharmacology.

[114]  Masahiko Watanabe,et al.  Synaptic distribution of the NR1, NR2A and NR2B subunits of the N‐methyl‐d‐aspartate receptor in the rat lumbar spinal cord revealed with an antigen‐unmasking technique , 2004, The European journal of neuroscience.

[115]  F. Nagy,et al.  Expression of vesicular glutamate transporters in rat lumbar spinal cord, with a note on dorsal root ganglia , 2004, The Journal of comparative neurology.

[116]  T. Hökfelt,et al.  Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles , 1997, Neuroscience.

[117]  S. Carlton,et al.  Peripheral glutamate release in the hindpaw following low and high intensity sciatic stimulation , 2000, Neuroreport.

[118]  J. Storm-Mathisen,et al.  Properties and localization of glutamate transporters. , 1998, Progress in brain research.