A simple method for electrical field stimulation of cultured granule cells.
A method suitable for electrical field stimulation of superfused primary cultures of cerebellar granule cells is described. A microchamber of about 0.5 ml was obtained by closing the culture dishes with a perspex plug equipped with stimulating electrodes and inlet-outlet tubing. Two-minute trains of electrical pulses (alternate polarity, 2-ms duration; 100 mA intensity; 10 V drop between electrodes; frequency 5, 10 and 20 Hz) applied to cultures kept at 27 degrees C, elicited a D-[3H]aspartate outflow which was frequency related, [Ca2+]0 dependent, tetrodotoxin sensitive. Moreover 2 trains of 10 Hz pulses (S1 and S2) at 30-min intervals caused an S2/S1 ratio equal or near to one, thus demonstrating that a steady-state condition had been achieved. The NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) but not the non-NMDA antagonist, 6-cyano-7-nitroquinozaline-2,3-dione (CNQX), added before S2, significantly increased the electrically evoked tritium efflux, suggesting that the endogenous transmitter released during electrical stimulation activated an NMDA-mediated negative feed-back. This technique of electrical field stimulation seems particularly feasible to study the extent and time course of drug effects on spontaneous and evoked D-[3H]aspartate outflow.
Department of Pharmacology, University of Ferrara, Italy.
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Bianchi
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Beani
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Antonelli
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Vedovato
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Calo
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Tomasini
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects; D004558:Electric Stimulation; D018691:Excitatory Amino Acid Antagonists; D018698:Glutamic Acid; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013779:Tetrodotoxin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D018691:Excitatory Amino Acid Antagonists; D011810:Quinoxalines; D001224:Aspartic Acid; D018698:Glutamic Acid; D013779:Tetrodotoxin; D011189:Potassium Chloride; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D014701:Veratridine; D015763:2-Amino-5-phosphonovalerate; D002118:Calcium