Role of the K⁺-Cl^– Cotransporter KCC2a Isoform in Mammalian Respiration at Birth

The KCC2a cotransporter is expressed in brainstem regions containing neuronal populations involved in respiration. A–C, Immunostainings against KCC2a (red) and ChAT (green) in transverse, 30-µm-thick brainstem slices obtained from a P0 KCC2a^+/+ animal at the axial level of the parafacial respiratory group (A), the preBötC (B), and the hypoglossus nucleus (C). Left: Schematic representations of isolated medullary preparations containing the pFRG (red shading) and the preBötC (orange shading). The violet horizontal line in each schematic indicates the rostro-caudal level corresponding to the images at right. The yellow rectangles in the middle overlay panels delimit the areas encompassing the pFRG, the preBötC, and the hypoglossus nucleus and are presented at a higher magnification in the three righthand panels in each case. Note that KCC2a is strongly expressed in these three neuronal groups that are all critically involved in generating respiration. VII, facial motor nucleus; nA, nucleus ambiguus; pFRG, parafacial respiratory group; XII, hypoglossus nucleus.

Rhythmogenesis by the isolated preBötC network is unaltered in KCC2a^–/– mutants at P0. A, Left: Schematic representation of a transverse brainstem slice preparation isolating the preBötC network (in orange) with the recording macroelectrode positioned on the slice surface to monitor spontaneous preBötC network activity. Right: Integrated recording traces of preBötC activity in a preparation obtained from a KCC2a^+/+ (black trace) and a KCC2a^–/– (blue trace) newborn mouse at P0. Note the absence of fictive inspiratory pauses in the mutant. B, Histograms representing individual mean frequency values (open circles) and the corresponding mean (± SEM) values for preBötC network activity measured in KCC2a^+/+ (n = 13; unfilled bar) and KCC2a^–/– (n = 17; blue bar) preparations. The inspiratory burst frequencies expressed by the preBötC network are comparable in the two genotypes. C, Immunostainings against KCC2a (red) and KCC2b (green) in transverse brainstem slices obtained from a P0 KCC2a^+/+ (top) and KCC2a^–/– (bottom) animal at the axial level of the preBötC. Note the coexpression of both isoforms in the WT and the complete absence of KCC2a in the mutant (the slight background labeling is substantially below the positive labeling obtained in preparations from wild-type animals and thus does not correspond to any significant immunoreactivity).

Respiratory rhythmogenesis by the isolated preBötC and pFRG networks is unaltered in KCC2a^–/– mutants at P0. A, Top left: Schematic representation of an isolated medullary preparation containing the pFRG (red shading) and preBötC (orange shading) networks with a suction electrode placed on a phrenic rootlet (C4) to record spontaneous rhythmic respiratory activity. The dashed green line indicates the sectioning level used to obtain the image at top right. Top right: Sagittal slice of a hindbrain preparation with the localization of motoneuronal groups (from ChAT immunostaining in green) demarking the rostral margin of the preparation. Bottom: Recordings of raw (top) and integrated (bottom) C4 activity in preparations obtained from a wild-type (upper black traces) and a KCC2a^–/– (lower blue traces) newborn mouse. Note the absence of fictive apneas in the mutant preparation. B, Histograms showing the overall mean (± SEM) cycle frequency values measured from 11 KCC2a^+/+ (unfilled bar) and 13 KCC2a^–/– (blue bar) isolated medullary preparations, together with mean values for individual preparations (open circles) obtained over a 3-min recording period. The fictive respiratory rhythm rate was not significantly different in KCC2a^–/– mutant preparations compared to wild-type preparations at P0. C, Histograms of cycle period distributions for 197 respiratory bursts measured in a KCC2a^+/+ preparation (left, unfilled bars) and 241 respiratory bursts in a KCC2a^–/– preparation (right, blue bars). No significant differences in the interburst interval distributions are evident, further indicating the similarity between that the rhythmic respiratory activities in isolated medullary preparations from the two genotypes.

Hypoglossus motor output is normal in KCC2a^–/– mutants at P0. A, Left: Schematic representation of an isolated medullary preparation containing both the pFRG (red shading) and preBötC (orange shading) networks with macroelectrodes placed on ventral rootlets to record rhythmic respiratory activity from the phrenic (C4) and hypoglossal (XII) nerves simultaneously. Right: Synchronized activity (indicated by dashed vertical lines) seen in integrated traces recorded from C4 (Int C4, bottom trace) and hypoglossal (Int XII, top trace) nerves in a KCC2a^–/– preparation at P0. B, Cross-correlation histogram showing perfect 1:1 synchronized bursting in the C4 and the XII rootlets of 7 different mutant preparations. The complete absence of any skipped cycles at the level of the hypoglossal root indicated that apneas observed in vivo are not due to a dysfunction of the upper airways motor command.

The extrinsic inhibitory influence from the pons is stronger in KCC2a^–/– mutants at P0. A, Top left: Schematic representation of an in vitro pontomedullary preparation containing the pontine structures and the pFRG (red shading) and preBötC (orange shading) networks with a macroelectrode placed on a phrenic rootlet (C4) to record rhythmic respiratory activity. The dashed green line indicates the level of sectioning for the image at right. Top right: Sagittal slice of a pontomedullary preparation showing the position of motoneuronal groups (ChaT immunoreactive populations in green) such as in the trigeminal nucleus (V), the facial nucleus (VII), and the nucleus ambiguus (nA). The black dashed line indicates the border between the pons and the medulla (also corresponding to the anterior boundary of isolated medullary preparations as shown in Fig. 3). Lower panel: Integrated traces of C4 activity (Int C4) in control conditions (top traces), in the presence of 1 µM SP (middle traces) and after application of the α2-AR antagonist yohimbine (50 µM; bottom traces) in KCC2a^+/+ (black traces at left) and KCC2a^–/– (blue traces at right) pontomedullary preparations at P0. Respiratory activity was completely absent in the mutant in control conditions but could be triggered by application of the excitatory neuromodulator SP or the α2-AR antagonist yohimbine (50 µM). B, Histograms depicting mean respiratory cycle frequency values (± SEM) obtained in KCC2a^+/+ (unfilled bars) and KCC2a^–/– (blue bars) pontomedullary preparations in control conditions, in the presence of 1 µM SP and in the presence of 50 µM yohimbine. C, Same layout as in B for measurements obtained from isolated medullary preparations (i.e., without the pons). Asterisks indicate statistical differences (p < 0.001). The number of experiments in each condition is indicated in the corresponding column.