Extracellular chloride and the maintenance of spontaneous epileptiform activity in rat hippocampal slices

Previous studies showed that furosemide blocks spontaneous epileptiform act ivity without diminishing synaptic transmission or reducing hyperexcited fi eld responses to electrical stimuli. We now test the hypothesis that the an tiepileptic effects of furosemide are mediated through its blockade of the Na+,K+,2Cl(-) cotransporter and thus should be mimicked by a reduction of e xtracellular chloride ([Cl-](o)). In the first set of experiments, field re cordings from the CA1 cell body layer of hippocampal slices showed that spo ntaneous bursting developed within 10-20 min in slices perfused with low-[C l-](o) (7 mM) medium but that this spontaneous epileptiform activity ceased after a further 10-20 min. Intracellular recordings from CA1 pyramidal cel ls showed that normal action potential discharge could be elicited by membr ane depolarization, even after the tissue was perfused with low-[Cl-](o) me dium for >2 h. In a second set of experiments, spontaneous bursting activit y was induced in slices by perfusion with high-[K+](o) (10 mM), bicuculline (100 mu M), or 4-aminopyridine (100 mu M). In each case, recordings from t he CA1 region showed that reduction of [Cl-](o) to 21 mM reversibly blocked the bursting within 1 h. Similar to previous observations with furosemide treatment, low-[Cl-](o) medium blocked spontaneous hypersynchronous dischar ges without reducing synaptic hyperexcitability (i.e., hyperexcitable field responses evoked by electrical stimulation). In a third set of experiments , prolonged exposure (>1 h after spontaneous bursting ceased) of slices to systematically varied [Cl-](o) and [K+](o) resulted in one of three types o f events: 1) spontaneous, long-lasting, and repetitive negative field poten tial shifts (7 mM [Cl-](o); 3 mM [K+](o)); 2) oscillations consisting of 5- to 10-mV negative shifts in the field potential, with a period of similar to 1 cycle/40 s (16 mM [Cl-](o); 12 mM [K+](o)); and 3) shorter, infrequent ly occurring negative field shifts lasting 20-40 s (21 mM [Cl-](o); 3 mM [K +](o)). Our observations indicate that the effects of low [Cl-](o) on neuro nal synchronization and spontaneous discharge are time dependent. Similar e ffects were seen with furosemide and low [Cl-](o), consistent with the hypo thesis that the antiepileptic effect of furosemide is mediated by the drug' s effect on chloride transporters. Finally, the results of altering extrace llular potassium along with chloride suggest that blockade of the Na+, K+,2 Cl(-) cotransporter, which normally transports chloride from the extracellu lar space into glial cells, is key to these antiepileptic effects.