Measurements with fluorescent probes in primary neural cultures; improved multiwell techniques.
BACKGROUND
Fluorescence imaging techniques are valuable tools for the pharmacological characterization of CNS drugs. Dissected cerebellar granule neurons (CGN) are an important model system in the study of mechanisms of excitotoxicity, glutamate receptors and transporters. Widely applied techniques use fluorescent probes loaded in neural cells cultured on glass supports. CGN, however, require at least 7 days for differentiation and over time cells tend to cluster and loose adherence to the glass substrate. This problem is accentuated in small wells (e.g. 96-well plates).


METHODS
CGN were grown on large coverslips (60 x 24 mm) and measurements made with a designed mountable multiwell in 48 regions on 4 coverslips at a time. The UV ratiometric probe fura-2 was used to measure glutamatergic calcium ([Ca(2+)](i)) responses induced by NMDA. The IC(50) of NMDA receptor antagonists was determined from inhibition curves with 6 doses and 8 parallels per experiment.


RESULTS
The method was validated by comparing with published data for the dose response to NMDA and glycine and IC(50) values for ion-channel block by Mg(2+) and MK-801.


CONCLUSIONS
Resolution is enhanced with the new technique since it allows measurement of multiple doses on cells from the same batch. It has advantages to cuvette techniques because cells have intact dendritic tree and synaptic function and it is a convenient method to obtain reliable dose-response curves for NMDA channel modulators on differentiated neural cells.
Ring
Avi
A
Department of Protection, Norwegian Defence Research Establishment, Kjeller, Norway. avi.ring@ffi.no
Tanso
Rita
R
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D016257:Fura-2 / Q000737:chemistry; D005998:Glycine / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011352:Procyclidine / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D002217:Carbachol; D011352:Procyclidine; D002118:Calcium; D005998:Glycine; D016257:Fura-2