Elsevier

Neuroscience

Volume 69, Issue 4, December 1995, Pages 1031-1055
Neuroscience

High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus

https://doi.org/10.1016/0306-4522(95)00350-RGet rights and content

Abstract

The cellular and subcellular localization of the G1uRA, G1uRB/C and G1uRD subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869–881), conjugated to carrier protein and adsorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of [H]kainate binding activity. The antibodies also recognize the G1uRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope.

All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the G1uRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the s stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the G1uRD subunit.

At the subcellular level, silver intensified immunogold particles for the GluRA, G1uRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CAI and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type I synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata.

The results demonstrate that the G1uRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Reference (130)

  • GulyasA.I. et al.

    Calretinin is present in non-pyramidal cells of the rat hippocampus—I. A new type of neuron specifically associated with the mossy fibre system

    Neuroscience

    (1992)
  • HerbA. et al.

    The KA-2 subunit of excitatory amino acid receptor shows widespread expression in brain and forms ion channels with distantly related subunits

    Neuron

    (1992)
  • HestrinS.

    Different glutamate receptor channels mediate fast excitatory synaptic currents in inhibitory and excitatory cortical neurons

    Neuron

    (1993)
  • HestrinS. et al.

    Mechanisms generating the time course of dual component excitatory synaptic currents recorded in hippocampal slices

    Neuron

    (1990)
  • JonasP. et al.

    Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression

    Neuron

    (1994)
  • JonesK.A. et al.

    Both NMDA and non-NMDA subtypes of glutamate receptors are concentrated at synapses on cerebral cortical neurons in culture

    Neuron

    (1991)
  • KatzL.C. et al.

    Scanning laser photostimulation: a new approach for analyzing brain circuits

    J. Neurosci. Meth.

    (1994)
  • KullmannD.M.

    Amplitude fluctuations of dual-component EPSCs in hippocampal pyramidal cells: implications for long-term potentiation

    Neuron

    (1994)
  • KuriyamaH. et al.

    Immunocytochemical localization of AMPA selective glutamate receptor subunits in the rat cochlea

    Hearing Res.

    (1994)
  • LiaoD. et al.

    Direct measurement of quantal changes underlying long-term potentiation in CA1 hippocampus

    Neuron

    (1992)
  • LivseyC.T. et al.

    Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons

    Neuron

    (1992)
  • LowryO.H. et al.

    Protein measurement by the Folin phenolreagent

    J. biol. Chem.

    (1951)
  • MartinL.J. et al.

    AMPA glutamate receptor subunits are differentially distributed in rat brain

    Neuroscience

    (1993)
  • MartinL.J. et al.

    Cellular localization of a metabotropic glutamate receptor in rat brain

    Neuron

    (1992)
  • MolnarE. et al.

    Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit. Cellular and subcellular distribution in the rat forebrain

    Neuroscience

    (1993)
  • MonaghanD.T. et al.

    Distribution of [3H]AMPA binding sites in rat brain as determined by quantitative autoradiography

    Brain Res.

    (1984)
  • MonyerH. et al.

    Developmental and regional expression in the rat brain and functional properties of four NMDA receptors

    Neuron

    (1994)
  • NakanishiN. et al.

    A family of glutamate receptor genes-evidence for the formation of heteromultimeric receptors with distinct channel proeprties

    Neuron

    (1990)
  • NusserZ. et al.

    Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization

    Neuroscience

    (1994)
  • PowD.V. et al.

    Extremely high titre polyclonal antisera against small neurotransmitter molecules: rapid production, characterization and use in light- and electron-microscopic immunocytochemistry

    J. Neurosci. Meth.

    (1993)
  • RaymondL.A. et al.

    Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity

    Trends Neurosci.

    (1993)
  • AmaralD.G.

    A Golgi study of cell types in the hilar region of the hippocampus in the rat

    J. comp. Neurol.

    (1978)
  • AndersenP. et al.

    Location and identification of excitatory synapses on hippocampal pyramidal cells

    Expl Brain Res.

    (1966)
  • AniksztejnL. et al.

    Quisqualate metabotropic receptors modulate NMDA currents and facilitate induction of long-term potentiation through protein kinase C

    Eur. J. Neurosci.

    (1992)
  • BaudeA. et al.

    Synaptic and nonsynaptic localization of the GluR1 subunit of the AMPA-type excitatory amino acid receptor in the rat cerebellum

    J. Neurosci.

    (1994)
  • BaudeA. et al.

    Cellular and subcellular localization of ionotropic (GluR1) and metabotropic (mGluRlα) glutamate receptors in rat hippocampus

    Soc. Neurosci. Abstr.

    (1993)
  • BaudeA. et al.

    The metabotropic glutamate receptor (mGluRl α) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction

    Neuron

    (1993)
  • BeesleyJ.E.

    Colloidal gold: A new perspective for cytochemical marking

  • BekkersJ.M. et al.

    NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus

    Nature

    (1989)
  • Ben-AriY. et al.

    Protein kinase C modulation of NMDA currents: an important link for LTP induction

    Trends Neurosci.

    (1992)
  • BenkeT.A. et al.

    N-Methyl-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons

  • BlackstoneC.D. et al.

    Biochemical characterization and localization of a non- N-methyl- d-aspartate glutamate receptor in rat brain

    J. Neurochem.

    (1992)
  • BlackstoneC. et al.

    Cyclic AMP and synaptic activity-dependent phosphorylation of AMPA-preferring glutamate receptors

    J. Neurosci.

    (1994)
  • BlackstadT.W. et al.

    Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers

    J. comp. Neurol.

    (1961)
  • BoulterJ. et al.

    Molecular cloning and functional expression of glutamate receptor subunit genes

    Science

    (1990)
  • BuhlE.H. et al.

    Diverse sources of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites

    Nature

    (1994)
  • BuzsakiG. et al.

    Direct afferent excitation and long-term potentiation of hippocampal interneurons

    J. Neurophysiol.

    (1982)
  • ClementsJ.D. et al.

    The time course of glutamate in the synaptic cleft

    Science

    (1992)
  • ColbertC.M. et al.

    Electrophysiological and pharmacological characterization of perforant path synapses in CA1: mediation by glutamate receptor

    J. Neurophysiol.

    (1992)
  • CollingridgeG.L. et al.

    Synaptic activation of N-methyl- d-aspartate receptors in the Schaffer collateral-commissural pathway of rat hippocampus

    J. Physiol.

    (1988)
  • Cited by (0)

    Present address: CNRS, Laboratoire de Neurobiologie, 31 chemin Joseph Aiguier, 13402 Marseille Cx20, France.

    View full text