Abstract
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.
Highlights
Primary dissociated neuronal cultures remain an important mainstay of neuroscience research
In dissociated hippocampal cultures tested at day in vitro 12–15, we found that routine media switches with fresh, supplementfree Neurobasal resulted in significant cell death
We were surprised to find that incubation in fresh Neurobasal medium, in the absence of L-glutamine or other supplements, killed mature hippocampal neurons
Summary
Primary dissociated neuronal cultures remain an important mainstay of neuroscience research. Various media have been optimized for neuronal survival and development in cell culture. Its use has extended well beyond its original optimization for survival of embryonic hippocampal neurons over a few days in cell culture [1]. Despite these extended uses, the effects of Neurobasal on most aspects of cell development remain incompletely characterized. In dissociated hippocampal cultures tested at day in vitro 12–15, we found that routine media switches (feedings) with fresh, supplementfree Neurobasal resulted in significant cell death. We conclude that NMDA agonist activity of Neurobasal medium should be taken into account in its use in neuronal cell culture
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
