Skip to main content

Advertisement

SpringerLink
Log in

Modelling of amyloid β-peptide induced lesions using roller-drum incubation of hippocampal slice cultures from neonatal rats

  • Research Article
  • Published:
Experimental Brain Research Aims and scope Submit manuscript

Abstract

Pronounced neurodegeneration of hippocampal pyramidal neurons has been shown in Alzheimer’s disease. The aim of this study was to establish an organotypic in vitro model for investigating effects of the amyloid β (Aβ)-peptide on pyramidal neuron degeneration, glial cell activation and tau phosphorylation. Tissue cultures in a quasi-monolayer were obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Neuronal populations identified included N-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons, and neurons immunopositive for glutamic acid decarboxylase-65 (GAD65) or gamma amino butyric acid (GABA). Many neurons expressed phosphorylated tau as shown by pS396, AD2 and PHF-tau immunostaining. Astrocytes, microglial cells and macrophages were also identified. The Aβ25–35 peptide formed fibrillar networks within 2 days as demonstrated by electron microscopy. In the presence of the neurotoxic Aβ25–35 peptide, but not Aβ35–25, deposits developed in the tissue that were stainable with Thioflavine T and Congo red and showed the characteristic birefringence of Aβ plaques. Following Aβ25–35 exposure, neurodegenerative cells were observed with Fluoro-Jade B staining. Further characterization of pyramidal neurons immunopositive for NMDA-R1 showed a decrease of cell number in the immediate surrounding of Aβ25–35 deposits in a time- and concentration-dependent fashion. Similar effects on pyramidal neurons were obtained following exposure to the full-length, Aβ1–40 peptide. Also, a loss of neuronal processes was seen with GAD65, but not GABA, immunohistochemistry after exposure to Aβ25–35. Aβ25–35-exposed neurons immunopositive for phospho-tau showed degenerating, bent and often fragmented processes. Astrocytes showed increased GFAP-positive reactivity after Aβ25–35 exposure and formation of large networks of processes. No obvious effect on microglial cells and macrophages could be seen after the Aβ25–35 exposure. The developed in vitro system may constitute a useful tool for screening novel drugs against Aβ-induced alterations of tau and degeneration of hippocampal neurons.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Abe K, Saito H (2000) Amyloid beta neurotoxicity not mediated by the mitogen-activated protein kinase cascade in cultured rat hippocampal and cortical neurons. Neurosci Lett 292:1–4

    Article  PubMed  CAS  Google Scholar 

  • Allen YS, Devanathan PH, Owen GP (1995) Neurotoxicity of beta-amyloid protein: cytochemical changes and apoptotic cell death investigated in organotypic cultures. Clin Exp Pharmacol Physiol 22:370–371

    Article  PubMed  CAS  Google Scholar 

  • Andersson H, Luthman J, Olson L (1994) Trimethyltin-induced expression of GABA and vimentin immunoreactivities in astrocytes of the rat brain. Glia 11:378–382

    Article  PubMed  CAS  Google Scholar 

  • Bamberger ME, Landreth GE (2002) Inflammation, apoptosis, and Alzheimer’s disease. Neuroscientist 8:276–283

    PubMed  CAS  Google Scholar 

  • Beach RL, Bathgate SL, Cotman CW (1982) Identification of cell types in rat hippocampal slices maintained in organotypic cultures. Brain Res 255:3–20

    PubMed  CAS  Google Scholar 

  • Bobinski M, Wegiel J, Tarnawski M, de Leon MJ, Reisberg B, Miller DC, Wisniewski HM, (1998) Duration of neurofibrillary changes in the hippocampal pyramidal neurons. Brain Res 799:156–158

    Article  PubMed  CAS  Google Scholar 

  • Braak H, Braak E, (1994) Morphological criteria for the recognition of Alzheimer’s disease and the distribution pattern of cortical changes related to this disorder. Neurobiol Aging 15:355–356, 379–380

    Google Scholar 

  • Buee-Scherrer V, Condamines O, Mourton-Gilles C, Jakes R, Goedert M, Pau B, Delacourte A (1996) AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer’s disease. Brain Res Mol Brain Res 39:79–88

    Article  PubMed  CAS  Google Scholar 

  • Burack MA, Halpain S (1996) Site-specific regulation of Alzheimer-like tau phosphorylation in living neurons. Neuroscience 72:167–184

    Article  PubMed  CAS  Google Scholar 

  • Busciglio J, Lorenzo A, Yeh J, Yankner BA (1995) beta-amyloid fibrils induce tau phosphorylation and loss of microtubule binding. Neuron 14:879–888

    Article  PubMed  CAS  Google Scholar 

  • Casal C, Serratosa J, Tusell JM (2002) Relationship between beta-AP peptide aggregation and microglial activation. Brain Res 928:76–84

    Article  PubMed  CAS  Google Scholar 

  • Combs CK, Coleman PD, O’Banion MK (1998) Developmental regulation and PKC dependence of Alzheimer’s-type tau phosphorylations in cultured fetal rat hippocampal neurons. Brain Res Dev Brain Res 107:143–158

    PubMed  CAS  Google Scholar 

  • Doig AJ, Hughes E, Burke RM, Su TJ, Heenan RK, Lu J (2002) Inhibition of toxicity and protofibril formation in the amyloid-beta peptide beta(25–35) using N-methylated derivatives. Biochem Soc Trans 30:537–542

    Article  PubMed  CAS  Google Scholar 

  • Domenici MR, Paradisi S, Sacchetti B, Gaudi S, Balduzzi M, Bernardo A, Ajmone-Cat MA, Minghetti L, Malchiodi-Albedi F (2002) The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures. J Physiol Paris 96:313–316

    Article  PubMed  CAS  Google Scholar 

  • Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, Houser CR (1994) Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 14:1834–1855

    PubMed  CAS  Google Scholar 

  • Gähwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4:329–342

    Article  PubMed  Google Scholar 

  • Giulian D, Haverkamp LJ, Yu JH, Karshin W, Tom D, Li J, Kirkpatrick J, Kuo LM, Roher AE (1996) Specific domains of beta-amyloid from Alzheimer plaque elicit neuron killing in human microglia. J Neurosci 16:6021–6037

    PubMed  CAS  Google Scholar 

  • Glenner GG (1989) The pathobiology of Alzheimer’s disease. Annu Rev Med 40:45–51

    Article  PubMed  CAS  Google Scholar 

  • Glenner GG, Wong CW (1984) Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885–890

    Article  PubMed  CAS  Google Scholar 

  • Goedert M, Jakes R, Crowther RA, Six J, Lubke U, Vandermeeren M, Cras P, Trojanowski JQ, Lee VM (1993) The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development. Proc Natl Acad Sci USA 90:5066–5070

    Article  PubMed  CAS  Google Scholar 

  • Goedert M, Jakes R, Vanmechelen E (1995) Monoclonal antibody AT8 recognizes tau protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett 189:167–170

    Article  PubMed  CAS  Google Scholar 

  • Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS, Wisniewski HM (1986) Microtubule associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem 261:6084–6089

    PubMed  CAS  Google Scholar 

  • Hanger DP, Betts JC, Loviny TLF, Blackstock WP, Anderton BH (1998) New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer’s disease brain using nanoelectronspray mass spectrometry. J Neurochem 71:2465–2476

    Article  PubMed  CAS  Google Scholar 

  • Harrigan MR, Kunkel DD, Nguyen LB, Malouf AT (1995) Beta amyloid is neurotoxic in hippocampal slice cultures. Neurobiol Aging 16:779–789

    Article  PubMed  CAS  Google Scholar 

  • Ikonomovic MD, Mizukami K, Warde D, Sheffield R, Hamilton R, Wenthold RJ, Armstrong DM (1999) Distribution of glutamate receptor subunit NMDAR1 in the hippocampus of normal elderly and patients with Alzheimer’s disease. Exp Neurol 160:194–204

    Article  PubMed  CAS  Google Scholar 

  • Klunk WE, Pettegrew JW, Abraham DJ (1989) Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem 37:1273–1281

    PubMed  CAS  Google Scholar 

  • Klunk WE, Wang Y, Huang GF, Debnath ML, Holt DP, Mathis CA (2001) Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 69:1471–1484

    Article  PubMed  CAS  Google Scholar 

  • LeVine H III (1993) Thioflavine T interaction with synthetic Alzheimer’s disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 2:404–410

    Article  PubMed  CAS  Google Scholar 

  • Luthman J, Radesäter AC, Öberg C (1998) Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1 beta- and LPS-induced hippocampal pyramidal cell loss in vitro. Amino Acids 14:263–269

    Article  PubMed  CAS  Google Scholar 

  • Malouf AT (1992) Effect of beta amyloid peptides on neurons in hippocampal slice cultures. Neurobiol Aging 13:543–551

    Article  PubMed  CAS  Google Scholar 

  • Milligan CE, Cunningham TJ, Levitt P (1991) Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J Comp Neurol 314:125–135

    Article  PubMed  CAS  Google Scholar 

  • Misonou H, Morishima-Kawashima M, Ihara Y (2000) Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells. Biochemistry 39:6951–6959

    Article  PubMed  CAS  Google Scholar 

  • Otvos L Jr, Feiner L, Lang E, Szendrei GI, Goedert M, Lee VM (1994) Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404. J Neurosci Res 39:669–673

    Article  PubMed  CAS  Google Scholar 

  • Pakaski M, Farkas Z, Kasa P Jr, Forgon M, Papp H, Zarandi M, Penke B, Kasa P Sr (1998) Vulnerability of small GABAergic neurons to human beta-amyloid pentapeptide. Brain Res 796:239–246

    Article  PubMed  CAS  Google Scholar 

  • Perlmutter LS, Barron E, Chui HC (1990) Morphologic association between microglia and senile plaque amyloid in Alzheimer’s disease. Neurosci Lett 119:32–36

    Article  PubMed  CAS  Google Scholar 

  • Pike CJ, Cotman CW (1993) Cultured GABA-immunoreactive neurons are resistant to toxicity induced by beta-amyloid. Neuroscience 56:269–274

    Article  PubMed  CAS  Google Scholar 

  • Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW (1991) In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res 563:311–314

    Article  PubMed  CAS  Google Scholar 

  • Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:1676–1687

    PubMed  CAS  Google Scholar 

  • Pike CJ, Cummings BJ, Cotman CW (1995a) Early association of reactive astrocytes with senile plaques in Alzheimer’s disease. Exp Neurol 132:172–179

    Article  CAS  Google Scholar 

  • Pike CJ, Walencewicz-Wasserman AJ, Kosmoski J, Cribbs DH, Glabe CG, Cotman CW (1995b) Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25–35 region to aggregation and neurotoxicity. J Neurochem 64:253–265

    Article  CAS  Google Scholar 

  • Rapoport M, Dawson HN, Binder LI, Vitek MP, Ferreira A (2002) Tau is essential to beta -amyloid-induced neurotoxicity. Proc Natl Acad Sci USA 99:6364–6369

    Article  PubMed  CAS  Google Scholar 

  • Sage BH Jr, O’Connell JP, Mercolino TJ (1983) A rapid, vital staining procedure for flow cytometric analysis of human reticulocytes. Cytometry 4:222–227

    Article  PubMed  Google Scholar 

  • Schmued LC, Hopkins KJ (2000) Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874(2):123–130

    Article  PubMed  CAS  Google Scholar 

  • Schmued LC, Albertson C, Slikker W Jr (1997) Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751:37–46

    Article  PubMed  CAS  Google Scholar 

  • Selkoe DJ, Yamazaki T, Citron M, Podlisny MB, Koo EH, Teplow DB, Haass C (1996) The role of APP processing and trafficking pathways in the formation of amyloid beta-protein. Ann N Y Acad Sci 777:57–64

    Article  PubMed  CAS  Google Scholar 

  • Sperk G, Schwarzer C, Tsunashima K, Fuchs K, Sieghart W (1997) GABA A receptor subunits in the rat hippocampus I: Immunocytochemical distribution of 13 subunits. Neuroscience 80:987–1000

    Article  PubMed  CAS  Google Scholar 

  • Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173–182

    Article  PubMed  CAS  Google Scholar 

  • Takashima A, Honda T, Yasutake K, Michel G, Murayama O, Murayama M, Ishiguro K, Yamaguchi H (1998) Activation of tau protein kinase I/glycogen synthase kinase-3beta by amyloid beta peptide (25–35) enhances phosphorylation of tau in hippocampal neurons. Neurosci Res 31:317–323

    Article  PubMed  CAS  Google Scholar 

  • Toro VC, Tehranian R, Zetterstrom M, Eriksson G, Langel U, Bartfai T, Iverfeld K (2001) Increased gene expression of interleukin-1alpha and interleukin-6 in rat primary glial cells induced by beta-amyloid fragment. J Mol Neurosci 17:341–350

    Article  PubMed  CAS  Google Scholar 

  • Yankner BA, Duffy LK, Kirschner DA (1990) Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250:279–282

    Article  PubMed  CAS  Google Scholar 

  • Zheng WH, Bastianetto S, Mennicken F, Ma W, Kar S (2002) Amyloid beta peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures. Neuroscience 115:201–211

    Article  PubMed  CAS  Google Scholar 

  • Zimmer J, Gähwiler BH (1984) Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata. J Comp Neurol 228:432–446

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Swedish Research Council (06537), the Swedish Heart and Lung Foundation, Alzheimerfonden, Gun and Bertil Stohnes stiftelse and Stiftelsen för Gamla Tjänarinnor.

Author information

Authors and Affiliations

  1. Local Discovery Research Area CNS& Pain Control, AstraZeneca, S-151 85, Södertälje, Sweden

    Sara Johansson, Ann-Cathrin Radesäter & Johan Luthman

  2. Karolinska Institutet, Neurotec Department, Division of Experimental Geriatrics, Novum, S-14186, Huddinge, Sweden

    Sara Johansson & Richard F. Cowburn

  3. Karolinska Institutet, Department of Cell and Molecular Biology, Medical Nobel Institute, Stockholm, Sweden

    Johan Thyberg

Authors
  1. Sara Johansson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ann-Cathrin Radesäter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard F. Cowburn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johan Thyberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johan Luthman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sara Johansson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johansson, S., Radesäter, AC., Cowburn, R.F. et al. Modelling of amyloid β-peptide induced lesions using roller-drum incubation of hippocampal slice cultures from neonatal rats. Exp Brain Res 168, 11–24 (2006). https://doi.org/10.1007/s00221-005-0069-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00221-005-0069-z

Keywords

Search

Navigation