Measurement of intracellular Ca2+ in cultured rat embryonic hippocampal neurons using a fluorescence microplate reader: potential application to biomolecular screening.
BACKGROUND
Fluorescence microplate readers for the measurement of cytosolic free Ca(2+) ([Ca(2+)](i)) are used as a drug screening tool, particularly for immortal cell lines. However, wider application of this methodological approach to more differentiated cells such as neurons would also be useful for the screening of compounds that modulate synaptic transmission. Such an approach has the potential to identify lead compounds for the development of novel drugs for the treatment of epilepsy, pathological pain states, Parkinson's disease, or other neurological disorders.


METHODS
In this paper, we describe the development of a microplate reader assay for the assessment of [Ca(2+)](i) in a primary culture of rat hippocampal neurons maintained in Neurobasal medium using the fluorescent calcium indicator, fluo-3.


RESULTS
The assay was appropriate for the screening of glutamate receptor agonists and antagonists. Furthermore, lowering the extracellular Mg(2+) concentration ([Mg(2+)](O)) produced consistent oscillations in neuronal [Ca(2+)](i) detected using the fluorescence microplate reader. These oscillations were inhibited by the GABA(B) agonist, baclofen, and the NMDA receptor antagonist, LY274614.


CONCLUSIONS
Our results indicate that assessment of the inhibitory effects of agents on spontaneous [Ca(2+)](i) oscillations in neurons may be useful for the identification of agents that act on targets for which specific screening methods are not currently available, or those which act via a previously unknown pathway to inhibit synaptic transmission. This technique also has the potential to increase the productivity of experiments designed to characterize changes in [Ca(2+)](i) (including calcium oscillations) in cultured neurons.
Hemstapat
Kamondanai
K
School of Pharmacy, University of Queensland, St. Lucia Campus, Brisbane, Queensland, 4072, Australia.
Smith
Maree T
MT
Monteith
Gregory R
GR
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005333:Fetus / Q000166:cytology / Q000196:embryology; D018756:GABA Antagonists / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D018756:GABA Antagonists; D007546:Isoquinolines; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; C070935:LY 274614; C059715:Fluo-3; D001418:Baclofen; D002118:Calcium