Immunogold localization of AMPA and NMDA receptors in somatic sensory cortex of albino rat

We performed an electron microscopic study of S‐1 cortex by using postembedding immunogold histochemistry to examine the subcellular distribution of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors (assessed with an antibody recognizing the glutamate receptor 2 and 3 [GluR2 and GluR3] subunits) and to compare this distribution with that of N‐methyl‐d‐aspartate (NMDA) receptors (assessed with an antibody for the NR1 subunit). Both receptors were concentrated at active zones of asymmetric synapses, often directly apposed to presynaptic dense bodies. GluR2/3 showed a bias for long active zones, whereas short active zones expressed GluR2/3 at substantially lower levels; in contrast, labeling for NR1 was independent of synaptic size. Particle counts suggested that synaptic labeling was Poisson distributed and implied that the majority of synapses express both receptors. Quantitative analysis indicates that approximately one‐half of synapses express high levels of GluR2/3 and that the remainder express GluR2/3 at a much lower level. Approximately three‐fourths of synapses express NR1 at a uniform level; the remainder, which may lack NR1 completely, include synapses with especially large active zones. The present results suggest that the smallest active zones may play a special role in synaptic plasticity. J. Comp. Neurol. 412:292–302, 1999. © 1999 Wiley‐Liss, Inc.

[1]  E. V. Van Bockstaele,et al.  Selective distribution of the NMDA‐R1 glutamate receptor in astrocytes and presynaptic axon terminals in the nucleus locus coeruleus of the rat brain: An immunoelectron microscopic study , 1996, The Journal of comparative neurology.

[2]  R. Huganir,et al.  Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments , 1998, The Journal of Neuroscience.

[3]  R. Silver,et al.  Non‐NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites. , 1996, The Journal of physiology.

[4]  Dr. Gareth Griffiths Fine Structure Immunocytochemistry , 1993, Springer Berlin Heidelberg.

[5]  R. Wenthold,et al.  Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain. , 1992, The Journal of biological chemistry.

[6]  R. Weinberg,et al.  Glutamate in thalamic fibers terminating in layer IV of primary sensory cortex , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[7]  R. Petralia,et al.  Variations in the tangential distribution of postsynaptic glutamate receptors in Purkinje cell parallel and climbing fiber synapses during development , 1998, Neuropharmacology.

[8]  R. Weinberg,et al.  Tangential synaptic distribution of NMDA and AMPA receptors in rat neocortex , 1997, Neuroscience Letters.

[9]  B. Sakmann,et al.  Fast and slow components of unitary EPSCs on stellate cells elicited by focal stimulation in slices of rat visual cortex. , 1992, The Journal of physiology.

[10]  R. Weinberg,et al.  An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[11]  P. Somogyi,et al.  Cellular, Subcellular, and Subsynaptic Distribution of AMPA-Type Glutamate Receptor Subunits in the Neostriatum of the Rat , 1997, The Journal of Neuroscience.

[12]  R. Huganir,et al.  Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain , 1993, Nature.

[13]  M. Giustetto,et al.  Glutamate receptors in the olfactory bulb synaptic circuitry: heterogeneity and synaptic localization of N-methyl-d-aspartate receptor subunit 1 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit 1 , 1996, Neuroscience.

[14]  T. Soderling,et al.  Differential Surface Expression and Phosphorylation of the N-Methyl-D-aspartate Receptor Subunits NR1 and NR2 in Cultured Hippocampal Neurons* , 1997, The Journal of Biological Chemistry.

[15]  Peter Somogyi,et al.  Cell Type and Pathway Dependence of Synaptic AMPA Receptor Number and Variability in the Hippocampus , 1998, Neuron.

[16]  B. Sakmann,et al.  Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression , 1994, Neuron.

[17]  F. Conti,et al.  Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes , 1996, Glia.

[18]  Mark Farrant,et al.  Differences in Synaptic GABAA Receptor Number Underlie Variation in GABA Mini Amplitude , 1997, Neuron.

[19]  O. Ottersen,et al.  Organization of AMPA Receptor Subunits at a Glutamate Synapse: A Quantitative Immunogold Analysis of Hair Cell Synapses in the Rat Organ of Corti , 1996, The Journal of Neuroscience.

[20]  T W Berger,et al.  Novel expression mechanism for synaptic potentiation: alignment of presynaptic release site and postsynaptic receptor. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  T. Dawson,et al.  Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[22]  R. Weinberg,et al.  Enhanced expression of AMPA receptor protein at perforated axospinous synapses , 1998, Neuroreport.

[23]  J. Bolam,et al.  Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat , 1998, The Journal of comparative neurology.

[24]  Tadaharu Tsumoto,et al.  Excitatory amino acid transmitters and their receptors in neural circuits of the cerebral neocortex , 1990, Neuroscience Research.

[25]  V. Gallo,et al.  Excitatory amino acid receptors in glia: Different subtypes for distinct functions? , 1995, Journal of neuroscience research.

[26]  R. Dingledine,et al.  The glutamate receptor ion channels. , 1999, Pharmacological reviews.

[27]  Mark Ellisman,et al.  Ultrastructural Localization of Dendritic Messenger RNA in Adult Rat Hippocampus , 1996, The Journal of Neuroscience.

[28]  P. Somogyi,et al.  Salient features of synaptic organisation in the cerebral cortex 1 Published on the World Wide Web on 3 March 1998. 1 , 1998, Brain Research Reviews.

[29]  H. Shida A study of protein A-gold resolution for immunoelectron microscopy. , 1991, Journal of electron microscopy technique.

[30]  R. Petralia,et al.  Ionotropic and metabotropic glutamate receptors show unique postsynaptic, presynaptic, and glial localizations in the dorsal cochlear nucleus , 1996, The Journal of comparative neurology.

[31]  G. Westbrook,et al.  Slow excitatory postsynaptic currents mediated by N‐methyl‐D‐aspartate receptors on cultured mouse central neurones. , 1988, The Journal of physiology.

[32]  H. Kamiya,et al.  Glutamate receptors in the mammalian central nervous system , 1998, Progress in Neurobiology.

[33]  R. Petralia,et al.  Immunocytochemistry of NMDA receptors. , 1999, Methods in molecular biology.

[34]  R. Nicoll,et al.  Silent Synapses Speak Up , 1997, Neuron.

[35]  R. Weinberg,et al.  GluR1‐immunopositive interneurons in rat neocortex , 1996, The Journal of comparative neurology.

[36]  T. Südhof,et al.  Binding of neuroligins to PSD-95. , 1997, Science.

[37]  J. Hablitz,et al.  EPSPs in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs. , 1989, Journal of neurophysiology.

[38]  V. Pickel,et al.  Comparative ultrastructural localization of the NMDAR1 glutamate receptor in the rat basolateral amygdala and bed nucleus of the stria terminalis , 1995, The Journal of comparative neurology.

[39]  L. Kaczmarek,et al.  Glutamate receptors in cortical plasticity: molecular and cellular biology. , 1997, Physiological reviews.

[40]  R. Huganir,et al.  The distribution of glutamate receptors in cultured rat hippocampal neurons: Postsynaptic clustering of AMPA selective subunits , 1993, Neuron.

[41]  S. Heinemann,et al.  Cloned glutamate receptors. , 1994, Annual review of neuroscience.

[42]  M. Bear,et al.  Common forms of synaptic plasticity in the hippocampus and neocortex in vitro. , 1993, Science.

[43]  T. Sato,et al.  A modified method for lead staining of thin sections. , 1968, Journal of electron microscopy.

[44]  R. Petralia,et al.  Light and electron immunocytochemical localization of AMPA‐selective glutamate receptors in the rat brain , 1992, The Journal of comparative neurology.

[45]  J. Eberwine,et al.  On the nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[46]  T. Salt,et al.  Thalamocortical and corticocortical excitatory postsynaptic potentials mediated by excitatory amino acid receptors in the cat motor cortexin vivo , 1995, Neuroscience.

[47]  P. Somogyi,et al.  High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus , 1995, Neuroscience.

[48]  R. Wenthold,et al.  Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[49]  J. Isaac,et al.  Evidence for silent synapses: Implications for the expression of LTP , 1995, Neuron.

[50]  J. Hablitz,et al.  Excitatory postsynaptic potentials in rat neocortical neurons in vitro. III. Effects of a quinoxalinedione non-NMDA receptor antagonist. , 1990, Journal of neurophysiology.

[51]  J. Morrison,et al.  Synaptic Distribution of the AMPA-GluR2 Subunit and Its Colocalization with Calcium-Binding Proteins in Rat Cerebral Cortex: An Immunohistochemical Study Using a GluR2-Specific Monoclonal Antibody , 1996, Experimental Neurology.

[52]  P. Somogyi,et al.  Synaptic and nonsynaptic localization of the GluR1 subunit of the AMPA- type excitatory amino acid receptor in the rat cerebellum , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[53]  R. Malinow,et al.  Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice , 1995, Nature.

[54]  R. Huganir,et al.  Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[55]  E. Michaelis Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging , 1998, Progress in Neurobiology.

[56]  P. Somogyi,et al.  Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit: Cellular and subcellular distribution in the rat forebrain , 1993, Neuroscience.

[57]  C. Stevens,et al.  NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus , 1989, Nature.

[58]  R. Huganir,et al.  AMPA glutamate receptor subunits are differentially distributed in rat brain , 1993, Neuroscience.

[59]  O. Ottersen,et al.  Organization of Glutamate Receptors at the Synapse , 1997, The European journal of neuroscience.

[60]  J. Pierce,et al.  An ultrastructural size principle , 1994, Neuroscience.

[61]  T. Bliss,et al.  A synaptic model of memory: long-term potentiation in the hippocampus , 1993, Nature.

[62]  W. Zieglgänsberger,et al.  The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain. , 1995, Brain research. Molecular brain research.

[63]  B. Clark,et al.  A Direct Comparison of the Single-Channel Properties of Synaptic and Extrasynaptic NMDA Receptors , 1997, The Journal of Neuroscience.

[64]  J. Morrison,et al.  Distribution of the excitatory amino acid receptor subunits GluR2(4) in monkey hippocampus and colocalization with subunits GluR5-7 and NMDAR1 , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[65]  R. Huganir,et al.  Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. , 1995, Science.

[66]  A. Thomson,et al.  A local circuit neocortical synapse that operates via both NMDA and non‐NMDA receptors , 1989, British journal of pharmacology.

[67]  R. Dingledine,et al.  Heterogeneity of synaptic glutamate receptors on CA3 stratum radiatum interneurones of rat hippocampus. , 1993, The Journal of physiology.

[68]  Eduard Kellenberger,et al.  1 – Some Basic Concepts for the Choice of Methods , 1991 .

[69]  P. Somogyi,et al.  Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization , 1994, Neuroscience.

[70]  R. Nicoll,et al.  Postsynaptically Silent Synapses in Single Neuron Cultures , 1998, Neuron.

[71]  M. Giustetto,et al.  Glutamate receptors in the olfactory bulb synaptic circuitry: heterogeneity and synaptic localization of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1. , 1997, Neuroscience.

[72]  R. Weinberg,et al.  Expression of NR2 receptor subunit in rat somatic sensory cortex: Synaptic distribution and colocalization with NR1 and PSD‐95 , 1999, The Journal of comparative neurology.

[73]  P. Courtoy,et al.  Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens. , 1983, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[74]  Fang Liu,et al.  Glutamate-mediated astrocyte–neuron signalling , 1994, Nature.

[75]  K. A. Jones,et al.  Both NMDA and non-NMDA subtypes of glutamate receptors are concentrated at synapses on cerebral cortical neurons in culture , 1991, Neuron.

[76]  J. Morrison,et al.  Synaptic coexistence of AMPA and NMDA receptors in the rat hippocampus: A postembedding immunogold study , 1998, Journal of neuroscience research.

[77]  R. Weinberg,et al.  AMPA Receptor Subunits Underlying Terminals of Fine-Caliber Primary Afferent Fibers , 1996, The Journal of Neuroscience.

[78]  Raymond Dingledine,et al.  Topology profile for a glutamate receptor: Three transmembrane domains and a channel-lining reentrant membrane loop , 1995, Neuron.

[79]  J. Morrison,et al.  Regional, cellular, and ultrastructural distribution of N-methyl-D-aspartate receptor subunit 1 in monkey hippocampus. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[80]  N W Daw,et al.  The role of NMDA receptors in information processing. , 1993, Annual review of neuroscience.

[81]  R. Wenthold,et al.  Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[82]  G Tocco,et al.  Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[83]  J. Morrison,et al.  Differential Subcellular Regulation of NMDAR1 Protein and mRNA in Dendrites of Dentate Gyrus Granule Cells after Perforant Path Transection , 1997, The Journal of Neuroscience.

[84]  T. Hicks,et al.  Excitatory amino acids and the cerebral cortex , 1996 .

[85]  J. Bolam,et al.  Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co‐localization at synapses with the GluR2/3 subunit of the AMPA receptor , 1998, The European journal of neuroscience.

[86]  R. Petralia,et al.  Endbulb Synapses in the Anteroventral Cochlear Nucleus Express a Specific Subset of AMPA-Type Glutamate Receptor Subunits , 1998, The Journal of Neuroscience.

[87]  E. Welker,et al.  The contribution of NMDA and non-NMDA receptors to fast and slow transmission of sensory information in the rat SI barrel cortex , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[88]  O. Ottersen,et al.  Differential Localization of δ Glutamate Receptors in the Rat Cerebellum: Coexpression with AMPA Receptors in Parallel Fiber–Spine Synapses and Absence from Climbing Fiber–Spine Synapses , 1997, The Journal of Neuroscience.

[89]  J. Morrison,et al.  Selective RNA editing and subunit assembly of native glutamate receptors , 1994, Neuron.

[90]  W. Janssen,et al.  Distribution and synaptic localization of immunocytochemically identified NMDA receptor subunit proteins in sensory-motor and visual cortices of monkey and human , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[91]  G. Collingridge,et al.  N-Methyl-D-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[92]  R. Weinberg,et al.  EM colocalization of AMPA and NMDA receptor subunits at synapses in rat cerebral cortex , 1996, Neuroscience Letters.

[93]  Joseph E LeDoux,et al.  Differential localization of NMDA and AMPA receptor subunits in the lateral and basal nuclei of the amygdala: A light and electron microscopic study , 1995, The Journal of comparative neurology.

[94]  B. Sakmann,et al.  Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS , 1995, Neuron.

[95]  Michael Hollmann,et al.  N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1 , 1994, Neuron.

[96]  M. Bennett,et al.  Alternatively spliced isoforms of the NMDARI receptor subunit , 1995, Trends in Neurosciences.

[97]  P. Streit Glutamate and Aspartate as Transmitter Candidates for Systems of the Cerebral Cortex , 1984 .

[98]  B Sakmann,et al.  Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices. , 1992, The Journal of physiology.