Excitotoxicity Triggered by Neurobasal Culture Medium
Open accessAbstract
Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12-15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca²⁺ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Glutamate was left out to avoid its excitotoxic effects in dissociated cultures.
“Glutamate was omitted to avoid the excitotoxicity of this amino-acid in dissociated cultures 47.”
From Efficient derivation of microglia-like cells from human pluripotent stem cells (2016) · cites this paper as [47]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones
Mayer, M. L., Westbrook, G. L., & Guthrie, P. B. (1984) · Nature · doi:10.1038/309261a0 · Cited by 2,906 · see citing works
The Role Of Glutamate Neurotoxicity In Hypoxic-Ischemic Neuronal Death
Choi, D. W., & Rothman, S. M. (1990) · Annual Review of Neuroscience · doi:10.1146/annurev.ne.13.030190.001131 · Cited by 2,210 · see citing works
Brewer, G. J., Torricelli, J. R., Evege, E. K., & Price, P. J. (1993) · Journal of Neuroscience Research · doi:10.1002/jnr.490350513 · Cited by 2,094 · see citing works
The Science of Stroke: Mechanisms in Search of Treatments
Moskowitz, M. A., Lo, E. H., & Iadecola, C. (2010) · Neuron · doi:10.1016/j.neuron.2010.07.002 · Cited by 1,930 · see citing works
Ionic dependence of glutamate neurotoxicity
Choi, D. W. (1987) · Journal of Neuroscience · doi:10.1523/jneurosci.07-02-00369.1987 · Cited by 1,854 · see citing works
Pharmacological agents acting at subtypes of metabotropic glutamate receptors
Schoepp, D. D., Jane, D. E., & Monn, J. A. (1999) · Neuropharmacology · doi:10.1016/s0028-3908(99)00092-1 · Cited by 1,061 · see citing works
Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death
Rothman, S. M. (1984) · Journal of Neuroscience · doi:10.1523/jneurosci.04-07-01884.1984 · Cited by 884 · see citing works
Long-term potentiation and functional synapse induction in developing hippocampus
Durand, G. M., Kovalchuk, Y., & Konnerth, A. (1996) · Nature · doi:10.1038/381071a0 · Cited by 789 · see citing works
Cerebral preconditioning and ischaemic tolerance
Gidday, J. M. (2006) · Nature Reviews Neuroscience · doi:10.1038/nrn1927 · Cited by 751 · see citing works
Goldberg, M. P., & Choi, D. W. (1993) · Journal of Neuroscience · doi:10.1523/jneurosci.13-08-03510.1993 · Cited by 693 · see citing works
Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability
Grubb, M. S., & Burrone, J. (2010) · Nature · doi:10.1038/nature09160 · Cited by 659 · see citing works
Patneau, D. K., & Mayer, M. (1990) · Journal of Neuroscience · doi:10.1523/jneurosci.10-07-02385.1990 · Cited by 653 · see citing works
The neurotoxicity of excitatory amino acids is produced by passive chloride influx
Rothman, S. (1985) · Journal of Neuroscience · doi:10.1523/jneurosci.05-06-01483.1985 · Cited by 636 · see citing works
Synaptic Desensitization of NMDA Receptors by Calcineurin
Tong, G., Shepherd, D., & Jahr, C. E. (1995) · Science · doi:10.1126/science.7878472 · Cited by 404 · see citing works
Mechanisms of L-Cysteine Neurotoxicity
Janáky, R., Varga, V., Hermann, A., Saransaari, P., & Oja, S. S. (2000) · Neurochemical Research · doi:10.1023/a:1007616817499 · Cited by 292 · see citing works
L-Cysteine, a Bicarbonate-Sensitive Endogenous Excitotoxin
Olney, J. W., Zorumski, C. F., Price, M. T., & Labruyere, J. (1990) · Science · doi:10.1126/science.2185543 · Cited by 257 · see citing works
Self-citationBeta- N -Methylamino-L-Alanine Neurotoxicity: Requirement for Bicarbonate as a Cofactor
Weiss, J. H., & Choi, D. W. (1988) · Science · doi:10.1126/science.3136549 · Cited by 203 · see citing works
Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures
Mennerick, S. J., Que, J., Benz, A. M., & Zorumski, C. F. (1995) · Journal of Neurophysiology · doi:10.1152/jn.1995.73.1.320 · Cited by 156 · see citing works
Self-citationHeterogeneity of sodium‐dependent excitatory amino uptake mechanisms in rat brain
Ferkany, J. W., & Coyle, J. T. (1986) · Journal of Neuroscience Research · doi:10.1002/jnr.490160305 · Cited by 132 · see citing works
Excitotoxicity, apoptosis and neuropsychiatric disorders
Olney, J. W. (2003) · Current Opinion in Pharmacology · doi:10.1016/s1471-4892(02)00002-4 · Cited by 130 · see citing works
Neural Activity and Survival in the Developing Nervous System
Zorumski, C. F., & Mennerick, S. J. (2000) · Molecular Neurobiology · doi:10.1385/mn:22:1-3:041 · Cited by 126 · see citing works
Self-citationShanker, G., & Aschner, M. (2001) · Journal of Neuroscience Research · doi:10.1002/jnr.10066 · Cited by 118 · see citing works
Development of excitotoxicity in cultured hippocampal neurons
Peterson, C., Neal, J. H., & Cotman, C. W. (1989) · Developmental Brain Research · doi:10.1016/0165-3806(89)90075-8 · Cited by 104 · see citing works
Shi, Q., Savage, J. E., Hufeisen, S. J., Rauser, L., Grajkowska, E., Ernsberger, P. R., Wroblewski, J. T., Nadeau, J. H., & Roth, B. L. (2003) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1124/jpet.102.047092 · Cited by 102 · see citing works
Mattson, M. P., Wang, H., & Michaelis, E. K. (1991) · Brain Research · doi:10.1016/0006-8993(91)91740-r · Cited by 98 · see citing works
Brewer, L. D., Thibault, O., Staton, J., Thibault, V., Rogers, J. T., García-Ramos, G., Kraner, S., Landfield, P., & Porter, N. (2007) · Brain Research · doi:10.1016/j.brainres.2007.03.020 · Cited by 96 · see citing works
Monyer, H., Giffard, R. G., Hartley, D. M., et al. (1992) · Neuron · doi:10.1016/0896-6273(92)90211-u · Cited by 89 · see citing works
Excitotoxic neuronal damage and neuropsychiatric disorders
Zorumski, C. F., & Olney, J. W. (1993) · Pharmacology & Therapeutics · doi:10.1016/0163-7258(93)90043-d · Cited by 80 · see citing works
Self-citationZorumski, C. F., Mennerick, S. J., & Que, J. (1996) · The Journal of Physiology · doi:10.1113/jphysiol.1996.sp021506 · Cited by 68 · see citing works
Self-citationGustafsson, B., & Wigström, H. (1990) · Progress in brain research · doi:10.1016/s0079-6123(08)61252-2 · Cited by 64 · see citing works
Vesicle Pool Heterogeneity at Hippocampal Glutamate and GABA Synapses
Moulder, K. L., Jiang, X., Taylor, A., Shin, W., Gillis, K. D., & Mennerick, S. J. (2007) · Journal of Neuroscience · doi:10.1523/jneurosci.2803-07.2007 · Cited by 60 · see citing works
Self-citationLow concentrations of inhibit the induction of long-term potentiation in rat hippocampal slices
Izumi, Y., Clifford, D. B., & Zorumski, C. F. (1992) · Neuroscience Letters · doi:10.1016/0304-3940(92)90414-3 · Cited by 58 · see citing works
Self-citationBidirectional Modulation of Cystine/Glutamate Exchanger Activity in Cultured Cortical Astrocytes
Tang, X., & Kalivas, P. W. (2003) · Annals of the New York Academy of Sciences · doi:10.1196/annals.1300.056 · Cited by 58 · see citing works
Homeostatic Effects of Depolarization on Ca2+Influx, Synaptic Signaling, and Survival
Moulder, K. L., Cormier, R. J., Shute, A. A., Zorumski, C. F., & Mennerick, S. J. (2003) · Journal of Neuroscience · doi:10.1523/jneurosci.23-05-01825.2003 · Cited by 48 · see citing works
Self-citationA false transmitter at excitatory synapses
Pan, Z. Z., Tong, G., & Jahr, C. E. (1993) · Neuron · doi:10.1016/0896-6273(93)90273-t · Cited by 38 · see citing works
Carbamate Formation and the Neurotoxicity of L-α Amino Acids
Nunn, P. B., Davis, A. J., & O’Brien, P. (1991) · Science · doi:10.1126/science.1859531 · Cited by 35 · see citing works
Mennerick, S. J., & Zorumski, C. F. (1996) · The Journal of Physiology · doi:10.1113/jphysiol.1996.sp021154 · Cited by 29 · see citing works
Self-citationContributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells
Mathisen, G. A., Fonnum, F., & Paulsen, R. E. (1996) · Neurochemical Research · doi:10.1007/bf02531643 · Cited by 28 · see citing works
Presynaptic silencing is an endogenous neuroprotectant during excitotoxic insults
Hogins, J., Crawford, D. C., Jiang, X., & Mennerick, S. J. (2011) · Neurobiology of Disease · doi:10.1016/j.nbd.2011.05.005 · Cited by 25 · see citing works
Self-citationCroucher, M. J., Thomas, L. S., Ahmadi, H., Lawrence, V., & Harris, J. R. (2001) · British Journal of Pharmacology · doi:10.1038/sj.bjp.0704138 · Cited by 24 · see citing works
Crawford, D. C., Chang, C. Y., Hyrc, K. L., & Mennerick, S. J. (2011) · Journal of Neuroscience · doi:10.1523/jneurosci.4960-10.2011 · Cited by 19 · see citing works
Self-citationAstrocytes exert a pro-apoptotic effect on neurons in postnatal hippocampal cultures
Shute, A. A., Cormier, R. J., Moulder, K. L., Benz, A. M., Isenberg, K. E., Zorumski, C. F., & Mennerick, S. J. (2005) · Neuroscience · doi:10.1016/j.neuroscience.2004.11.025 · Cited by 13 · see citing works
Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
Efficient derivation of microglia-like cells from human pluripotent stem cells
Muffat, J., Li, Y., Yuan, B., Mitalipova, M., Omer, A., Corcoran, S., Bakiasi, G., Tsai, L.-H., Aubourg, P., Ransohoff, R. M., et al. (2016) · Nature Medicine · doi:10.1038/nm.4189 · Cited by 651 · see citing works
Neuronal culture microenvironments determine preferences in bioenergetic pathway use
Sünwoldt, J., Bosche, B., Meisel, A., & Mergenthaler, P. (2017) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2017.00305 · Cited by 34 · see citing works
- Authors
- Hogins, J., Crawford, D. C., Zorumski, C. F., & Mennerick, S.
- Affiliations
- Washington University in St. Louis (US)
- Corresponding author
- Mennerick, Steven
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2011-09-28
- Volume
- 6
- Issue
- 9
- Pages
- e25633
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3182245
- OpenAlex
- W2149959655
- Cited by
- 41
- References
- 42
- Citations by year
- 2025: 1 · 2022: 3 · 2021: 2 · 2020: 6 · 2019: 3 · 2018: 3 · 2017: 8 · 2016: 7 · 2015: 5 · 2014: 1 · 2012: 2
- FWCI
- 0.40
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Digital Commons@Becker (Washington University School of Medicine) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Institutes of Health (DA07261)
- National Institute on Alcohol Abuse and Alcoholism (R01AA017413, AA017413)
- National Institute of Mental Health (MH078823, MH07791, R01MH078823)
- National Institute on Drug Abuse (DA07261, T32DA007261)
- National Institute of Neurological Disorders and Stroke (F31NS066611, NS52823, NS066611)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Keywords
- excitotoxicity, NMDA receptor, cysteine, hippocampal neurons, neuronal cell death, D-APV, primary neuronal cultures, calcium influx
- MeSH terms
- Animals, Culture Media, Cysteine, Electric Conductivity, Female, Hippocampus, Male, Neurons, Neurotoxins, Receptors, N-Methyl-D-Aspartate, N-Methylaspartate, Cell Death, Cell Culture Techniques, Rats
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
