Elsevier

Brain Research

Volume 565, Issue 1, 22 November 1991, Pages 94-108
Brain Research

Research report
Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons

https://doi.org/10.1016/0006-8993(91)91740-RGet rights and content

Abstract

The mechanisms regulating the expression and localization of excitatory amino acid (EAA) neurotransmitter receptors in neurons of the developing mammalian brain, and roles for these receptors in the plasticity and degeneration of neural circuits are not well understood. We previously isolated and characterized a 71 kDa glutamate binding protein (GBP) from rat brain, and have recently obtained evidence that this GBP is a component of a functional N-methyl-d-aspartate (NMDA) receptor-ion channel complex. We have now used antibodies to this putative NMDA receptor protein to examine its expression and localization, and consequences of its activation in cultured embryonic (18 day) rat hippocampal neurons. Immunocytochemistry and Western blots using monoclonal antibodies to the GBP demonstrated an increase in GBP-positive neurons and their staining intensity with time in culture, GBP was localized to the somata and dendrites of pyramidal-like neurons and was sparse or absent in the axons. The expression and compartmentalization of GBP occurred in isolated neurons indicating that direct cell interactions were not required for these processes. Cell surface staining for GBP occurred in patches on the soma and dendrites. The developmental expression of GBP immunoreactivity closely paralleled the expression of sensitivity to NMDA neurotoxicity. There was a direct relationship between GBP immunoreactivity and neuronal vulnerability to glutamate-induced degeneration; vulnerable neurons stained heavily whereas resistant neurons showed either low levels of staining or no staining. Finally, a GBP antiserum greatly reduced NMDA neurotoxicity (but not kainate neurotoxicity). Taken together, these findings demonstrate the expression of presumptive NMDA receptors within a subpopulation of embryonic hippocampal neurons, and their segregation to the soma and dendrites of pyramidal neurons. This spatial distribution of glutamate receptors among and within neurons is likely to play important roles in regulating the structure of neural circuitry during development, and may also be an important determinant of selective neuronal vulnerability in pathological conditions.

References (47)

  • M.J. Eaton et al.

    Immunochemical characterization of brain synaptic membrane glutamate-binding proteins

    J. Biol. Chem.

    (1990)
  • J.T. Greenamyre et al.

    Excitatory amino acids and Alzheimer's disease

    Neurobiol. Aging

    (1989)
  • D.R. Hampson et al.

    A kainic acid receptor from frog brain purified using domoic acid affinity chromatography

    J. Biol. Chem.

    (1988)
  • D.R. Hampson et al.

    Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping

    J. Biol. Chem.

    (1989)
  • W.F. Maragos et al.

    Glutamate dysfunction in Alzheimer's disease: an hypothesis

    Trends Neurosci.

    (1987)
  • M.P. Mattson et al.

    Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry

    Neuron

    (1988)
  • M.P. Mattson et al.

    Isolated hippocampal neurons in cryopreserved long term culture: development of neuroarchitecture and sensitivity to NMDA

    Int. J. Dev. Neurosci.

    (1988)
  • M.P. Mattson

    Cellular signaling mechanisms common to the development and degeneration of neuroarchitecture

    Mech. Ageing Dev.

    (1989)
  • M.P. Mattson et al.

    Development and selective neurodegeneration in cell cultures from different hippocampal regions

    Brain Research

    (1989)
  • M.P. Mattson

    Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and calcium influx in cultured hippocampal neurons

    Neuron

    (1990)
  • J.W. McDonald et al.

    Physiological and pathophysiological roles of excitatory amino acids during central nervous system development

    Brain Res. Rev.

    (1990)
  • A. Represa et al.

    Development of high affinity kainate binding sites in human and rat hippocampi

    Brain Research

    (1986)
  • A. Represa et al.

    Transient increase of NMDA-binding sites in human hippocampus during development

    Neurosci. Lett.

    (1989)
  • Cited by (100)

    • Blockers of adenosine A1, but not muscarinic acetylcholine, receptors improve excessive extracellular glutamate-induced synaptic depression

      2013, Neuroscience Research
      Citation Excerpt :

      Co-application of AP-5 similarly increased the recoveries in PSs and fEPSPs after the washout, indicating that NMDA-type glutamate receptors contribute to the excessive glutamate-induced excitatory synaptic depression but do not significantly work to depress somatic excitability. Selective depression of dendritic excitatory synaptic transmissions without that of somatic excitability, mediated by NMDA-type glutamate receptors localizing on both dendrites and a soma in a hippocampal pyramidal neuron (Mattson et al., 1991), is a novel finding, which can be observed by simultaneous PS and fEPSP recordings with the MEA system. Immediate recovery of edrophonium-induced fEPSP depression after its washout and lack of an effect of edrophonium on PSs indicate that its ChE inhibiting action does not remain after the washout, at which time excessive glutamate-induced synaptic change was evaluated in the present study.

    • Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl-d-asparate-type glutamate receptors

      2010, Neuroscience
      Citation Excerpt :

      However, significant methodological differences between these studies are apparent, most notably, the age of tissue in vitro (5 vs. 10 days in vitro, after Mulholland and Prendergast, 2003 and Ikegaya and Matsuki, 2002, respectively). Previous work employing dissociated hippocampi has demonstrated that in vitro aging (as early as 0–8 days in culture) is associated with increased sensitivity of hippocampal pyramidal cells to NMDA-induced excitability (Mattson et al., 1991). Thus, the differences described above with regard to the necessity of tri-synaptic hippocampal circuitry for the expression of CA1 pyramidal cell injury may be related to the in vitro aging.

    • Immediate neuronal preconditioning by NS1619

      2009, Brain Research
      Citation Excerpt :

      Positive immunostaining for microtubule-associated protein-2 and negative immunostaining for glial fibrillary acidic protein verified that the cultures were composed of more than 98% of neurons on day 7 in vitro. Experiments were carried out on 7–9-day old cultures, during which period neurons expressed NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate, and kainate receptors and were vulnerable to glucose deprivation (Mattson et al., 1991, 1993). For medium changes, fresh FM was used in all experiments.

    • Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures

      2009, Brain Research
      Citation Excerpt :

      Positive immunostaining for microtubule-associated protein-2 and negative immunostaining for glial fibrillary acidic protein verified that the cultures were composed of more than 98% of neurons on day 7 in vitro (DIV 7). Experiments were carried out on 7–9 day old cultures, during which neurons expressed N-methyl-d-aspartate, α-amino-3-hydroxy-5-methylisoxazole-4-propionate, and kainate receptors and were vulnerable to glucose deprivation (Mattson et al., 1991, 1993). Adenoviral vectors were stored in 3% sucrose/phosphate buffered saline (PBS) at − 80 °C.

    View all citing articles on Scopus
    View full text