Fig 1.
Histological analysis of parafacial regions.
A) Localization of injections into pFV and pFL. Transverse view of medulla at Bregma -11.25 mm. Red circles show locations of injection sites for pFV and pFL. Green dashed box is magnified in C. B) Ventral view of medullary surface with location of pFV and pFL injection sites, marked by white circles, superimposed. C) Micrographs of injection sites. Green marks staining for choline acetyl transferase (ChAT), highlighting the cholinergic neurons of the facial (VII) nucleus, and red marks fluorescent beads coinjected with micropipette solutions in to the pFV and pFL. Py–Pyramidal tract, SP-5 –Spinal trigeminal tract, 7n –Facial nucleus.
Table 1.
Statistical analysis.
Fig 2.
B+SpFL induces active expiration.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for B+SpFL. Bi) Rest. Bii) Following B+SpFL. Grey vertical boxes demark period of each breath taken up by inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). C) Comparison between ventilation at rest (Rst) and after B+SpFL injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or B+SpFL group. *: p < 0.05. frequency–f, TI−inspiratory period TE,–expiratory period, tidal volume–VT, GGEMG−genioglossus electromyogram, DiaEMG−diaphragm electromyogram, AbdEMG−abdominal electromyogram. BPM–breaths per minute, a.u.—arbitrary units.
Fig 3.
B+SpFV increases f, decreases VT, and induces post-inspiratory activity in abdominal muscles and pre- and post-inspiratory activity in gengioglossus muscles.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for B+SpFV. Examples of sighs are marked with arrowheads labelled with #. Post-inspiratory burst AbdEMGs are marked with arrowheads labelled with †. Bi) Rest. Bii) Following B+SpFV. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). Sigh marked by #. Post-Inspiratory AbdEMG marked by †. C) Comparison between ventilation at rest (Rst) and after B+SpFV injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or B+SpFL group. *: p < 0.05. Abbreviations defined in Fig 2.
Fig 4.
SalinepFL does not affect breathing.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for SalinepFL. Bi) Rest. Bii) Following SalinepFL. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). a.u.: arbitrary units. C) Comparison between ventilation at rest (Rst) and after SalinepFL. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or salinepFL group. Abbreviations defined in Fig 2.
Fig 5.
SalinepFV does not affect breathing.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for SalinepFV. Bi) Rest. Bii) Following SalinepFV. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). a.u.: arbitrary units. C) Comparison between ventilation at rest (Rst) and after SalinepFV. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or SalinepFV group. Abbreviations defined in Fig 2.
Table 2.
Median and interquartile range for all recorded variables.
Fig 6.
AMPApFL induces active expiration.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for AMPApFL. Bi) Rest. Bii) Following AMPApFL. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). C) Comparison between ventilation at rest (Rst) and after AMPApFL injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or AMPApFL group. *: p < 0.05. Abbreviations defined in Fig 2.
Fig 7.
AMPApFV increases f and VT, but does not induce post-inspiratory activity in either abdominal muscles or in pre- and post-inspiratory activity gengioglossus muscles.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for AMPApFV. Bi) Rest. Bii) Following AMPApFV. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). C) Comparison between ventilation at rest (Rst) and after AMPApFV injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or AMPApFV group. *: p < 0.05. Abbreviations defined in Fig 2.
Fig 8.
GlupFV alters, but does not induce, post-inspiratory activity in either abdominal muscles or in pre- and post-inspiratory activity in genioglossus muscles.
A) Integrated traces from a single experiment, gray arrows indicate unilateral injections for GlupFV. Ai) Vagus intact. Aii) Vagotomized. B) Comparison between ventilation at rest (Rst) and after GlupFV. Bi) Vagus intact. Bii) Vagotomized. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or GlupFV group. *: p < 0.05. Abbreviations defined in Fig 2.
Fig 9.
A+NpFL does not affect breathing.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for A+NpFL. Bi) Rest. Bii) Following A+NpFL. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). C) Comparison between ventilation at rest (Rst) and after A+NpFL injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or A+NpFL group. Abbreviations defined in Fig 2.
Fig 10.
A+NpFV decreases VT, and reduces output of inspiratory muscles.
A) Integrated traces from a single experiment. Black arrows at bottom indicate epochs in expanded traces (Bi and Bii), gray arrows at top indicate unilateral injections for A+NpFV. Bi) Rest. Bii) Following A+NpFV. Grey vertical boxes demark phases of each breath: inspiration (I; light gray), post-inspiration (Post: medium grey), and pre-inspiration (Pre: Dark gray). C) Comparison between ventilation at rest (Rst) and after A+NpFV injection. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for each parameter, i.e., f, TI, TE, VT, GGEMG, DiaEMG, or AbdEMG regardless of whether it belonged to control or A+NpFV group. *: p < 0.05. Abbreviations defined in Fig 2.
Fig 11.
Schematic of minimal bCPG, which consists of 4 essential components.
1) preBötzinger Complex (preBötC) drives inspiration by exciting inspiratory premotor neuronal populations projecting to inspiratory muscles, e.g., diaphragm and tongue, and inhibits pFL; 2) parafacial Dorsocaudal (pFDC) contains GRP positive neurons contributing to basal sigh rhythm. 3) pFL drives active expiration by exciting expiratory premotor neuronal populations projecting to expiratory muscles, e.g., abdominals and tongue, and excites neurons that inhibit preBötC, either in preBötC or in BötC (not shown); 4) pFV contains neurons and glia that contribute to CO2/pH regulation and integrates sensory afferents affecting breathing, including basal sigh rate, via excitatory connections to preBötC and breathing premotor and motor neurons. pFV contains 4 subpopulations: i) tonically active neurons that modulate VT and diaphragm bursting at rest; ii) tonically suppressed neurons that modulate f; iii) NMB positive neurons that affect basal sigh rate, and; iv) tonically suppressed neurons that provide rhythmic drive to abdominal and genioglossus expiratory motor pools producing active expiration.