Fig 1.
Quantification of Trp transcript expression in preBötC neurons.
Mean Trp mRNA expression in reads per kilobase of transcript per million mapped reads (RPKM) in Dbx1 and non-Dbx1 preBötC neurons from Dbx1;Ai9 mice at postnatal day 2, pseudocolor scale at right. Transcripts organized by relative expression levels in the Dbx1 neurons. Some of the Trp transcript data were displayed in a different format as supplemental information in [27]. Data are publicly available in the NCBI Gene Expression Omnibus database, https://www.ncbi.nlm.nih.gov/geo/, accession no. GSE100356. Dbx1, developing brain homeobox 1; NCBI, National Center for Biotechnology Information; preBötC, pre-Bötzinger complex; RPKM, reads per kilobase of transcript per million mapped reads; Trp, transient receptor potential.
Fig 2.
Trpm4 ion channels in preBötC neurons.
(A) Transverse section from an adult Dbx1;Ai9 mouse showing tdTomato (magenta) expression in Dbx1-derived cells. (B) Selection box from A encompasses the preBötC and shows Trpm4 expression (cyan). (C) Selection box from the preBötC in B showing tdTomato (Dbx1) and Trpm4 expression in preBötC neurons. Trpm4 is expressed in both Dbx1 neurons (arrowheads) and non-Dbx1 neurons (arrow). (D) GFAP expression (yellow) identifies Dbx1-derived astrocytes of the preBötC (yellow and magenta, marked with an asterisk), which lack Trpm4 expression. Arrowhead identifies a Dbx1 neuron with Trpm4 expression. Separate scale bars are shown for A, B, C, and D. Dbx1, developing brain homeobox 1; GFAP, glial fibrillary acidic protein; preBötC, pre-Bötzinger complex; Trp, transient receptor potential.
Fig 3.
Trpc3 ion channels in preBötC neurons.
(A) Transverse section from an adult Dbx1;Ai9 mouse showing tdTomato (magenta) expression in Dbx1-derived cells. (B) Selection box from A focuses on the preBötC core and shows tdTomato (Dbx1) and Trpc3 expression (cyan). Trpc3 is expressed in both Dbx1 neurons (arrowheads) and non-Dbx1 neurons (arrows). (C) GFAP expression (yellow) identifies Dbx1-derived astrocytes of the preBötC (yellow and magenta, marked with an asterisk), which lack Trpc3 expression. Arrowhead identifies a Dbx1 neuron with Trpc3 expression. Separate scale bars are shown for A, B, and C. Dbx1, developing brain homeobox 1; GFAP, glial fibrillary acidic protein; preBötC, pre-Bötzinger complex; Trp, transient receptor potential.
Table 1.
Quantification of transcript copies of Trp genes per 2.5 ng cDNA synthesized from RNA extracted from the preBötC of adult mice and measured by ddPCR.
Fig 4.
Breathing in an awake, intact adult mouse.
A 30 sec continuous sample of breathing via whole-body plethysmography in a CD-1 mouse. Inspiratory airflow points down. The trace shows exploratory movements (orange), grooming (green), and high-frequency sniffing (purple), in addition to eupnea (yellow). One sigh (gray, expanded in the inset) characterized by 2-fold larger inspiration compared to a typical eupneic breath, followed by expiration. Time and airflow calibrations are shown. Raw data can be found in the Supporting information (S1 Data).
Fig 5.
Longitudinal study of breathing in a Trpm4 shRNA-injected mouse.
(A) Breathing at different time points from before injection (baseline) until day 31 after injection of Trpm4 shRNA in an adult CD-1 mouse. Gasps on days 28 and 31 are recognized by rapid active exhale (upward trajectory before inspiration), followed by inhale (downward trajectory) efforts. This mouse died on day 32. (B,C) Group data showing tallies of gasps (B) and deaths (C) in mice injected with Trpm4 (cyan), Trpc3 (magenta), and nontargeting control (gray) shRNA. Elapsed time following shRNA injection was binned in 6-day increments. Primary data can be found in the Supporting information (S1 Data). shRNA, short hairpin RNA; Trp, transient receptor potential.
Fig 6.
Longitudinal study of breathing after shRNA injections in three mouse cohorts.
Breathing measurements in awake adult CD-1 mice after shRNA injection targeting Trpm4 (left, cyan), Trpc3 (middle, magenta), and a nontargeting control sequence (right, gray). Rows from top to bottom show VT, TI, frequency, and MV. Each animal was assigned a unique symbol (circle, square, triangle, hexagon, plus sign, asterisk, and variations on those symbols), which tracks the breathing measurements for that animal during the postinjection observation period. Best-fit lines and the 95% confidence intervals are shown. The number of points in the left column (Trpm4) decreases as animals died of respiratory failure starting at the bin for days 25–30. Primary data can be found in the Supporting information (S1 Data). shRNA, short hairpin RNA; Trp, transient receptor potential.
Fig 7.
Trpm4 but not Trpc3 antagonists attenuate preBötC rhythms in vitro.
(A) preBötC field potentials (black) and XII output (gray) in control and at steady state following approximately 20 min application of 100 μM 9-phenanthrol in neonatal CD-1 mice. A typical eupnea-related inspiratory burst is marked with ε; a typical sigh burst is marked with σ. (B–E) Area of inspiratory (B) and sigh (C) bursts as well as inspiratory (D) and sigh (E) frequency in control and 9-phenanthrol. (F) preBötC field potentials (black) and XII output (gray) in control and at steady state following approximately 20 min application of 10 μM pyr-3. Again, ε marks a eupnea-related inspiratory burst; σ marks a sigh burst. (G–J) Area of inspiratory (G) and sigh (H) bursts as well as inspiratory (I) and sigh (J) frequency in control and pyr-3. Mean ± SD shown (magenta) for all graphs in B–D and G–J. Primary data can be found in the Supporting information (S1 Data). preBötC, pre-Bötzinger complex; pyr-3, pyrazole-3; XII, hypoglossal nerve root.
Fig 8.
Trpm4 and Trpc3 antagonists attenuate inspiratory and sigh bursts of Dbx1 preBötC neurons.
Data are from neonatal Dbx1;Ai9 mice. Each row shows inspiratory (left) and sigh bursts (right) superimposed at the onset of whole-cell recording (t0, black) and at steady-state (ss, gray, following approximately 20 min of whole-cell dialysis or [in the case of pyr-3] bath application). Group data plotted at far right with means and SD bars (magenta). (A) no drug dialysis, (B) BAPTA, (C) FFA, (D) 9-phenanthrol, and (E) pyr-3. Primary data can be found in the Supporting information (S1 Data). BAPTA, (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid); Dbx1, developing brain homeobox 1; FFA, flufenamic acid; preBötC, pre-Bötzinger complex; pyr-3, pyrazole-3; Trp, transient receptor potential.