Treatment with furosemide indirectly increases inhibitory transmission in the developing hippocampus

During the first two postnatal weeks intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing γ-aminobutyric acid (GABA) responses. GABAergic depolarization in the immature brain is crucial for the formation and maturation of excitatory synapses, but when GABAergic signaling becomes inhibitory it no longer promotes synapse formation. Here we examined the role of chloride transporters in developing postnatal hippocampal neurons using furosemide, an inhibitor of the chloride importer NKCC1 and chloride exporter KCC2 with reported anticonvulsant effects. We treated organotypic hippocampal cultures made from 6 to 7-day old mice with 200 μM furosemide from DIV1 to DIV8. Using perforated patch clamp recordings we observed that the GABA reversal potential was depolarized after acute furosemide application, but after a week of furosemide treatment the GABA reversal potential but was more hyperpolarized compared to control. Expression levels of the chloride cotransporters were unaffected after one week furosemide treatment. This suggests that furosemide inhibited KCC2 acutely, while prolonged treatment resulted in (additional) inhibition of NKCC1, but we cannot exclude changes in HCO 3 - . We assessed the effects of accelerating the GABA shift by furosemide treatment on inhibitory synapses onto CA1 pyramidal cells. Directly after cessation of furosemide treatment at DIV9, inhibitory synapses were not affected. However at DIV21, two weeks after ending the treatment, we found that the frequency of inhibitory currents was increased, and VGAT puncta density in stratum Radiatum was increased. In addition, cell capacitance of CA1 pyramidal neurons was reduced in furosemide-treated slices at DIV21 in an activity-dependent manner. Our results suggest that furosemide treatment indirectly promoted inhibitory transmission, possibly by increasing activity-independent GABA release.