Culturing conditions determine neuronal and glial excitability.
The cultivation of pure neuronal cultures is considered advantageous for the investigation of cell-type specific responses (such as transmitter release and also pharmacological agents), however, divergent results are a likely consequence of media modifications and culture composition. Using Fura-2 based imaging techniques, we here set out to compare calcium responses of rat hippocampal neurones and glia to excitatory stimulation with l-glutamate in different culture types and media. Neurones in neurone-enriched cultures had increased responses to 10 μM and 100 μM l-glutamate (+43 and 45%, respectively; p's< 0.001) and a slower recovery compared to mixed cultures, indicating heightened excitability. In matured (15-20 days in vitro) mixed cultures, neuronal responder rates were suppressed in a neurone-supportive medium (Neurobasal-A, NB: 65%) compared to a general-purpose medium (supplemented minimal essential medium, MEM: 96%). Glial response size in contrast did not differ greatly in isolated or mixed cultures maintained in MEM, but responder rates were suppressed in both culture types in NB (e.g. 10 μM l-glutamate responders in mixed cultures: 29% in NB, 71% in MEM). This indicates that medium composition is more important for glial excitability than the presence of neurones, whereas the presence of glia has an important impact on neuronal excitability. Therefore, careful consideration of culturing conditions is crucial for interpretation and comparison of experimental results. Especially for investigations of toxicity and neuroprotection mixed cultures may be more physiologically relevant over isolated cultures as they comprise aspects of mutual influences between glia and neurones.
School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, Scotland, UK.
Sandra
S
Stoppelkamp
Gernot
G
Riedel
Bettina
B
Platt
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media; D018846:Excitatory Amino Acids / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D006706:Homeostasis / Q000502:physiology; D007150:Immunohistochemistry; D009457:Neuroglia / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D051381:Rats; D017470:Receptors, Glutamate / Q000502:physiology
D003470:Culture Media; D018846:Excitatory Amino Acids; D005456:Fluorescent Dyes; D018696:Neuroprotective Agents; D017470:Receptors, Glutamate; D018698:Glutamic Acid; D002118:Calcium; D016257:Fura-2