RHYTHM GENERATION IN ORGANOTYPIC MEDULLARY CULTURES OF NEWBORN RATS

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium cont ained rhythmically discharging neurones which were activated by CO2 an d H+. The mechanisms underlying this rhythmicity and the spread of exc itation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our fi ndings showed that (1) Exposure to tetrodotoxin (0.2 mu M) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished per iodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 mu M) and/or hydroxysaclofen (200 mu M) no r the blockade of glycinergic potentials with strychnine hydrochloride (100 mu M) abolished rhythmicity. (3) While atropine sulphate (5 mu M ) was ineffective in modulating periodic discharges nicotine (100 mu M ) like CO2- shortened the intervals between the periodic events; hexam ethonium (50-100 mu M) reduced both periodic and aperiodic activity. ( 4) Exposure to the NMDA antagonist 2-aminophosphone valeric acid (50 m u M) suppressed period ic events only transiently. In the presence of 2-aminophosphone valeric acid rhythmicity recovered. However, the AMPA -antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-50 mu M), abolish ed periodic activity reversibly within less than 5 min. When 6-cyano-7 -nitroquinoxaline-2,3-dione and nicotine were administered simultaneou sly periodic events persisted for up to 10 min. These findings indicat e that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate en dogenous cellular mechanisms which initiate oscillations of intracellu lar calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagon ists verapamil and flunarizine (50-100 mu M each) and the inorganic ca lcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed period ic activity and abolished or weakened the chemosensitivity towards CO2 /acidosis. (6) Dantrolene (10 mu M), an inhibitor of intracellular cal cium release decreased the periodicity, while thapsigargin (2 mu M) wh ich blocks endoplasmic Ca2+-ATPase, transiently accelerated the occurr ence of periodic events. (7) Oscillations of intracellular free calciu m concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)-(7) indicate that transmembrane c alcium fluxes as well as intracellular Ca2+-release and -clearance mec hanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in me dullary neurones.