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

Previous studies showed that furosemide blocks spontaneous epileptiform activity without diminishing synaptic transmission or reducing hyperexcited field responses to electrical stimuli. We now test the hypothesis that the antiepileptic effects of furosemide are mediated through its blockade of the Na+,K+,2Cl(-) cotransporter and thus should be mimicked by a reduction of extracellular chloride ([Cl-](o)). In the first set of experiments, field recordings from the CA1 cell body layer of hippocampal slices showed that spontaneous bursting developed within 10-20 min in slices perfused with low-[Cl-](o) (7 mM) medium but that this spontaneous epileptiform activity ceased after a further 10-20 min. Intracellular recordings from CA1 pyramidal cells showed that normal action potential discharge could be elicited by membrane depolarization, even after the tissue was perfused with low-[Cl-](o) medium for >2 h. In a second set of experiments, spontaneous bursting activity 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 the 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 discharges 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 of events: 1) spontaneous, long-lasting, and repetitive negative field potential 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, infrequently 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 neuronal synchronization and spontaneous discharge are time dependent. Similar effects were seen with furosemide and low [Cl-](o), consistent with the hypothesis that the antiepileptic effect of furosemide is mediated by the drug's effect on chloride transporters. Finally, the results of altering extracellular potassium along with chloride suggest that blockade of the Na+, K+,2Cl(-) cotransporter, which normally transports chloride from the extracellular space into glial cells, is key to these antiepileptic effects.