title: Addition of glutamate to serum-free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro.
abstract: A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25muM for 1-7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI).
authors.affiliation: NanoScience Technology Center, Orlando, FL 32826, USA.
authors.forename: Darin
authors.initials: D
authors.lastname: Edwards
authors.forename: Mainak
authors.initials: M
authors.lastname: Das
authors.forename: Peter
authors.initials: P
authors.lastname: Molnar
authors.forename: James J
authors.initials: JJ
authors.lastname: Hickman
mesh_terms: D000375:Aging; D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D016895:Culture Media, Serum-Free; D005110:Extracellular Space / Q000378:metabolism; D005898:Glass; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000502:physiology*; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000502:physiology; D009416:Nerve Regeneration; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D016895:Culture Media, Serum-Free; D018698:Glutamic Acid
references.citation: J Neurosci Methods. 1995 Nov;62(1-2):111-9
references.pmid: 8750092
references.citation: Hippocampus. 2008;18(3):281-93
references.pmid: 18058822
references.citation: J Neurosci Methods. 1997 Feb;71(2):143-55
references.pmid: 9128149
references.citation: Biotechnol Prog. 2003 Nov-Dec;19(6):1756-61
references.pmid: 14656152
references.citation: Stroke. 1998 Sep;29(9):1937-50; discussion 1950-1
references.pmid: 9731622
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14512-7
references.pmid: 12391303
references.citation: Cell. 2000 Dec 8;103(6):945-56
references.pmid: 11136979
references.citation: Nat Neurosci. 2000 Jul;3(7):661-9
references.pmid: 10862698
references.citation: Appl Microbiol Biotechnol. 2006 Apr;70(3):273-80
references.pmid: 16463172
references.citation: J Neurosci Res. 2005 Apr 1;80(1):104-13
references.pmid: 15744743
references.citation: Cell. 1992 Dec 11;71(6):973-85
references.pmid: 1458542
references.citation: Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11070-4
references.pmid: 7972011
references.citation: Exp Gerontol. 2005 May;40(5):426-37
references.pmid: 15919595
references.citation: Nat Rev Neurosci. 2003 Nov;4(11):885-900
references.pmid: 14595400
references.citation: Exp Neurol. 2008 Jan;209(1):171-80
references.pmid: 18005959
references.citation: Brain Res. 1993 Dec 10;630(1-2):136-47
references.pmid: 8118680
references.citation: In Vitro Cell Dev Biol Anim. 2005 Nov-Dec;41(10):343-8
references.pmid: 16448224
references.citation: J Neurosci. 1988 Jun;8(6):2087-100
references.pmid: 2898515
references.citation: Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11024-31
references.pmid: 11572963
references.citation: J Neurosci Methods. 1998 Aug 1;82(2):167-73
references.pmid: 9700689
references.citation: J Physiol. 2006 Apr 15;572(Pt 2):313-21
references.pmid: 16469782
references.citation: J Neurosci. 2002 Nov 15;22(22):9868-76
references.pmid: 12427843
references.citation: Curr Top Med Chem. 2006;6(10):961-8
references.pmid: 16787270
references.citation: Cell Mol Life Sci. 1999 Aug 30;55(11):1448-62
references.pmid: 10518992
references.citation: J Neurosci Methods. 1998 Jan 31;79(1):37-46
references.pmid: 9531458
references.citation: Brain Res. 1984 Apr;315(2):314-8
references.pmid: 6722593
references.citation: Am J Geriatr Psychiatry. 2005 Dec;13(12):1052-61
references.pmid: 16319297
references.citation: Neuron. 2002 Apr 11;34(2):221-33
references.pmid: 11970864
references.citation: Physiol Rev. 2008 Apr;88(2):769-840
references.pmid: 18391179
references.citation: J Neurosci Res. 1993 Aug 1;35(5):567-76
references.pmid: 8377226
references.citation: Trends Biotechnol. 2001 Aug;19(8):317-23
references.pmid: 11451474
references.citation: J Neurochem. 1966 Nov;13(11):1149-55
references.pmid: 5924663
references.citation: Neurobiol Aging. 1998 Nov-Dec;19(6):561-8
references.pmid: 10192215
references.citation: Prog Neurobiol. 1998 Aug;55(5):433-61
references.pmid: 9670213
references.citation: Brain Res. 1988 Apr-Jun;472(2):179-212
references.pmid: 2898278