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Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

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Abstract

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.

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References

  1. Hansson E, Thorlin T (1999) Brain primary cultures and vibrodissociated cells as tools for the study of astroglial properties and functions. Dev Neurosci 21:1–11

    Article  CAS  PubMed  Google Scholar 

  2. Hertz L, Zielke HR (2004) Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci 27:735–743

    Article  CAS  PubMed  Google Scholar 

  3. Danbolt NC (2001) Glutamate uptake. Progr Neurobiol 65:1–105

    Article  CAS  Google Scholar 

  4. Martinez-Hernandez A, Norenberg MD (1977) Glutamine synthetase: glial localization in brain. Science 195:1356–1358

    Article  CAS  PubMed  Google Scholar 

  5. Haydon PG, Carmignoto G (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 86:1009–1031

    Article  CAS  PubMed  Google Scholar 

  6. Muyderman H, Nilsson M, Blomstrand F, Khatibi S, Olsson T, Hansson E, Rönnbäck L (1998) Modulation of mechanically induced calcium waves in hippocampal astroglial cells inhibitory efffects of alpha 1-adrenergic stimulation. Brain Res 793:127–135

    Article  CAS  PubMed  Google Scholar 

  7. Judd MG, Nagaraja TN, Brookes N (1996) Potassium-induced stimulation of glutamate uptake in mouse cerebral astrocytes: the role of intracellular pH. J Neurochem 66:169–176

    Article  CAS  PubMed  Google Scholar 

  8. Trotti D, Nussberger S, Volterra A, Hediger MA (1997) Differential modulation of the uptake currents by redox interconversion of cysteine residues in the human neuronal glutamate transporter EAAC1. Eur J Neurosci 9:2207–2212

    Article  CAS  PubMed  Google Scholar 

  9. Rodriguez-Kern A, Gegelashvili M, Schousboe A, Zhang J, Sung L, Gegelashvili G (2003) Beta-amyloid and brain-derived neurotrophic factor, BDNF, up-regulate the expression of glutamate transporter GLT-1/EAAT2 via different signaling pathways utilizing transcription factor NF-kappaB. Neurochem Int 43:363–370

    Article  CAS  PubMed  Google Scholar 

  10. Brewer GJ (1995) Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res 42:674–683

    Article  CAS  PubMed  Google Scholar 

  11. Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J Neurosci Res 35:567–576

    Article  CAS  PubMed  Google Scholar 

  12. Hansson E, Rönnbäck L, Persson LI, Lowenthal A, Noppe M, Alling C, Karlsson B (1984) Cellular composition of primary primary cultures from cerebral cortex, striatum, hippocampus, brainstem and cerebellum. Brain Res 300:9–18

    Article  CAS  PubMed  Google Scholar 

  13. Persson M, Brantefjord M, Hansson E, Rönnbäck L (2005) Lipopolysaccharide increases microglial GLT-1 expression and glutamate uptake capacity in vitro by a mechanism dependent on TNF-alpha. GLIA 51:111–120

    Article  PubMed  Google Scholar 

  14. Leonova J, Thorlin T, Åberg ND, Eriksson PS, Rönnbäck L, Hansson E (2001) Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes. Am J Physiol Cell Physiol 281:C1495–C1503

    CAS  PubMed  Google Scholar 

  15. Hansson E, Eriksson P, Nilsson M (1985) Amino acid and monoamine transport in primary astroglial cultures from defined brain regions. Neurochem Res 10:1335–1341

    Article  CAS  PubMed  Google Scholar 

  16. Blomstrand F, Khatibi S, Muyderman H, Hansson E, Olsson T, Rönnbäck L (1999) 5-Hydroxytryptamine and glutamate modulate velocity and extent of intercellular calcium signalling in hippocampal astroglial cells in primary cultures. Neuroscience 88:1241–1253

    Article  CAS  PubMed  Google Scholar 

  17. Swanson RA, Liu J, Miller JW, Rothstein JD, Farrell K, Stein BA, Longuemare MC (1997) Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Neurosci 17:932–940

    CAS  PubMed  Google Scholar 

  18. Schlag BD, Vondrasek JR, Munir M, Kalandadze A, Zelenaia OA, Rothstein JD, Robinson MB (1998) Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. Mol Pharmacol 53:355–369

    CAS  PubMed  Google Scholar 

  19. Becher A, Drenckhahn A, Pahner I, Margittai M, Jahn R, Ahnert-Hilger G (1999) The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation. J Neurosci 19:1922–1931

    CAS  PubMed  Google Scholar 

  20. Ängehagen M, Ben-Menachem E, Shank R, Rönnbäck L, Hansson E (2004) Topiramate modulation of kainate-induced calcium currents is inversely related to channel phosphorylation level. J Neurochem 88:320–325

    Article  PubMed  Google Scholar 

  21. Guillet B, Lortet S, Masmejean F, Samuel D, Nieoullon A, Pisano P (2002) Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures. Neurochem Int 40:661–671

    Article  CAS  PubMed  Google Scholar 

  22. Perego C, Vanoni C, Bossi M, Massari S, Basudev H, Longhi R, Pietrini G (2000) The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal co-cultures. J Neurochem 75:1076–1084

    Article  CAS  PubMed  Google Scholar 

  23. Schluter K, Figiel M, Rozyczka J, Engele J (2002) CNS region-specific regulation of glial glutamate transporter expression. Eur J Neurosci 16:836–842

    Article  PubMed  Google Scholar 

  24. Zelenaia O, Schlag BD, Gochenauer GE, Ganel R, Song W, Beesley JS, Grinspan JB, Rothstein JD, Robinson MB (2000) Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol Pharmacol 57:667–678

    CAS  PubMed  Google Scholar 

  25. Kanai Y, Hediger MA (2004) The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch 447:469–479

    Article  CAS  PubMed  Google Scholar 

  26. Kugler P, Schleyer V (2004) Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus. Hippocampus 14:975–985

    Article  CAS  PubMed  Google Scholar 

  27. Hein C, Horvath E, Kugler P (2001) Glutamate transporter expression in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex. Eur J Neurosci 13:1839–1848

    Article  CAS  PubMed  Google Scholar 

  28. Kaneko T, Fujiyama F, Hioki H (2002) Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain. J Com Neurol 444:39–62

    Article  CAS  Google Scholar 

  29. Williams SM, Sullivan RK, Scott HL, Finkelstein DI, Colditz PB, Lingwood BE, Dodd PR, Pow DV (2005) Glial glutamate transporter expression patterns in brains from multiple mammalian species. GLIA 49:520–541

    Article  PubMed  Google Scholar 

  30. Raudensky J, Yamamoto BK (2007) Effects of chronic unpredictable stress and methamphetamine on hippocampal glutamate function. Brain Res 1135:129–135

    Article  CAS  PubMed  Google Scholar 

  31. Andersson AK, Rönnbäck L, Hansson E (2005) Lactate induces tumour necrosis factor-α, interleukin-6 and interleukin-1β release in microglial- and astroglia-enriched primary cultures. J Neurochem 93:1327–1333

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The research was supported by the Swedish Research Council (Grant no. 33X-06812), Edith Jacobson’s Foundation, and Folksams Forskingsstiftelse. The excellent technical assistance of Barbro Eriksson and Mona Brantefjord is gratefully acknowledged.

Conflict of interest

None of the authors have any conflict of interest.

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Authors and Affiliations

  1. Department of Clinical Neuroscience and Rehabilitation, the Sahlgrenska Academy, Institute of Neuroscience and Physiology, University of Gothenburg, Per Dubbsgatan 14, 1tr, 413 45, Gothenburg, Sweden

    Ulrika Björklund, Mikael Persson, Lars Rönnbäck & Elisabeth Hansson

Authors
  1. Ulrika Björklund
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  2. Mikael Persson
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  3. Lars Rönnbäck
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  4. Elisabeth Hansson
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Correspondence to Elisabeth Hansson.

Additional information

Ulrika Björklund and Mikael Persson contributed equally much to this work.

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Björklund, U., Persson, M., Rönnbäck, L. et al. Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons. Neurochem Res 35, 1733–1742 (2010). https://doi.org/10.1007/s11064-010-0236-x

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  • DOI: https://doi.org/10.1007/s11064-010-0236-x

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