Figure 5. Atoh1 neurons are necessary for biphasic cervical sigh-like fictive breaths.

E18.5 WT mice produce biphasic respiratory-related cervical bursts (C4, green) with lumbar (L1, magenta) activation only during the initial normal amplitude cervical burst under baseline (A) or in the presence of 1 µM SP (B). Arrow in B indicates likely active inhibition of lumbar root during the larger amplitude cervical burst peak. (C) Biphasic cervical bursts persist after loss of RTN neurons in Phox2b-Cre;Atoh1^LacZ/F (RL⁺/RTN⁻) mice with the lumbar burst occurring during the larger amplitude burst. Schematics indicate maintenance (top left) or targeted loss (right) of Phox2b RTN (purple) populations in WT or Phox2b-Cre;Atoh1^LacZ/F (RL⁺/RTN⁻) mice. (D–E) In contrast to lumbar respiratory output (magenta), XIth internal intercostal (D, IC, black), or VIIn (E, VII, dark blue) can occur during both the initial and larger amplitude cervical burst (green) in E18.5 WT (D) or control Atoh1^LacZ/+ heterozygote (E). Note the co-activation of cervical and VII roots during lumbar inhibition (E). (F–I) Atoh1^LacZ/LacZ (RL⁻/RTN⁻) mice do not exhibit biphasic respiratory cervical bursts. Schematic indicates targeted loss of Phox2b RTN (purple) and Atoh1 RL (orange) neurons. (F–G) Single integrated traces showing temporal separation between cervical (green) and lumbar (magenta) peaks due to increased noise (arrows). Numbers under traces indicate time of the lumbar peak in relation to the cervical peak (in ms). In some fictive breaths, Atoh1^LacZ/LacZ mice show respiratory doublets with two distinct cervical outputs with likely lumbar inhibition (arrow in I) during the initial burst in the presence of 1 µM SP (H) or 10⁻¹² M SST (I). Scale bar = 1 s. Figure 5—figure supplement 1 shows the variability in amplitude and pattern of biphasic cervical bursts other respiratory bursts during baseline rhythmic activity as well as average and overlapping standard and biphasic bursts in a E18.5 WT mouse preparation.

DOI: http://dx.doi.org/10.7554/eLife.02265.014

Figure 5—figure supplement 1. Normal and biphasic cervical respiratory bursts show different cervical and lumbar patterns and vary in amplitude during fictive breathing in an E185 mouse brainstem preparation.

(A) 100 s recording of integrated endogenous respiratory-related output from cervical (C4, green) and lumbar (L1, magenta) motor roots in an E18.5 WT mouse brainstem-spinal cord preparation. Letters above peaks (B–G) are expanded below for clarity. Note the variations in amplitude of both cervical and lumbar bursts. (B–G) 2 s overlapping traces of integrated cervical and lumbar motor output expanded from A. Peaks have been scaled to the same height for ease of comparison of the patterns of activity. Biphasic cervical bursts (B, F–G) show lumbar activity primarily during the initial cervical peak. Note that some fictive breaths that do not show biphasic cervical activity can have either smaller (C–D) or larger (E) amplitude motor output than biphasics bursts. Scale bar 10 s (A), 500 ms (B–G). (H) Integrated average top and overlapped cervical (middle, green) and lumbar (bottom, magenta) traces from 10 consecutive fictive breaths lacking biphasic cervical output. Note the variation in amplitude in cervical traces. Integrated average traces are scaled for clarity. (I) Integrated average top and overlapped cervical (middle, green) and lumbar (bottom, magenta) traces from 10 sequential, but not consecutive, fictive breaths showing biphasic cervical output.

DOI: http://dx.doi.org/10.7554/eLife.02265.015