(a) Disinhibition via local microinjection of picrotoxin (GABAA receptor antagonist) and strychnine (glycine receptor antagonist) into the ventral spinal cord at C3-C6, the location of the PMC, converts the highly patterned firing of phrenic MNs in control mice to the more continuous firing observed in Hoxa5MNΔ; c5het mice. (b) The percentage of time during inspiratory bursts with no unit activity is equivalent in both control +PTX/Strych and Hoxa5MNΔ; c5het mice, and is considerably reduced from control mice (Figure 6). (c–d) Power spectra and the percentage of phrenic MN unit firing above and below 75 Hz are equivalent in control +PTX/Strych and Hoxa5MNΔ; c5het mice. For relative power definition, see Materials and methods (n = 5 control +PTX/Strych, 8 Hoxa5MNΔ; c5het for b-d). (e) The number of perisomatic inhibitory synapses on phrenic MNs, identified by the apposition of GAD67 and gephyrin, is reduced in P10 Hoxa5MNΔ; c5het mice. A single phrenic MN cell body is shown and representative inhibitory synapses are labelled with stars. Choline Acetyltransferase (ChAT) staining was used to identify the cell body membrane. (f) Examples of inhibitory perisomatic synapses showing apposition of GAD67 and gephyrin. (g) Quantification of the reduction in perisomatic inhibitory synapses in Hoxa5MNΔ; c5het mice (n = 86 phrenic MNs from 2 control mice, 78 phrenic MNs from 2 Hoxa5MNΔ; c5het mice). Scale bar = 10 μm in e, 0.25 μm in f. See also Figure 7—figure supplement 1.