In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Clconcentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor

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... control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a -/mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves. The authors declare no competing financial interests. Author contributions: J.S., P.U., and M.T.-B. designed research; C.J.D., L.C., V.S., M.M., and M.T.-B. performed research and analyzed data; P.U., M.S.A., J.S., and M.T.-B. wrote the paper. Significance Statement The expression of the neuronal specific potassium chloride cotransporter KCC2 is necessary for the generation of respiratory activity at birth, but the specific role of the KCC2a isoform in this process remains unknown. By examining in vivo and in vitro the breathing motor output of newborn KCC2a -/mice, we observed a lower breathing frequency and a high occurrence of apneas at birth. These anomalies, expressed mainly at P0 (day of birth), do not arise from changes in the brainstem respiratory rhythmgenerating circuits, but appear to result from abnormally strong inhibitory pontine neuromodulatory influences that target these networks. Our results provide evidence for a transient but important role of the KCC2a isoform in the proper development of the central respiratory command.