TRPM4 mediates a subthreshold membrane potential oscillation in respiratory chemoreceptor neurons that drives pacemaker firing and breathing

Figure 1.. The properties of subthreshold oscillation in RTN neurons (A) Spontaneous action potentials recorded in RTN neurons under whole-cell current clamp were abolished by TTX (0.5 μM); traces shown on expanded timescale. In this and all subsequent experiments, the bath solution contained fast synaptic blockers (10 μM CNQX, 10 μM bicuculline, and 30 μM strychnine). (B) After TTX, depolarizing current injection (10 pA steps, from −100 mV) failed to elicit action potentials; active depolarizing potentials were superimposed on the passive response with larger current pulses (arrows). (C) In TTX, small membrane potential fluctuations at −50 mV were transformed into large subthreshold oscillations when DC was injected (to ~−40 mV). (D) Individual oscillations for a period of 30 s at −40 mV are overlaid (black) and averaged (red). Inset: Phase-plane plot of the averaged oscillation waveform. (A–D from the same neuron). (E and F) The nadir and peak (E) and amplitude (F) of averaged subthreshold oscillation waveforms (n = 75). (G and H) Subthreshold oscillations were obtained at the indicated membrane potentials by DC injection (G), and corresponding power density-frequency plots were…

Figure 2.. Ca2+- and Na+-dependent subthreshold oscillations involve TRPM4 channels in RTN neurons (A–F) Effects on subthreshold oscillations in TTX (0.5 μM TTX, at −40 mV with DC injection) of bath application of NMDG (A), substituted for Na+; Cd2+ (B), (100 μM); nifedipine (C), L-type Ca2+ channel blocker (at 10 μM); flufenamate (D), broad-spectrum TRP channel blocker (at 100 μM); and two TRPM4 channel blockers, 9-phenanthrol (E) (30 μM) and CBA (F) (50 μM). Summary data depict effects of each manipulation on integrated power (from FFT analysis) for all cells. Statistical analyses by paired t test: NMDG (p = 0.005, n = 7); Cd2+ (p = 0.003, n = 9); nifedipine (p = 0.007, n = 5); FFA (p < 0.0001, n = 10); 9-pt (p = 0.002, n = 15); CBA (p = 0.036, n = 8). Box and whiskers plots as in Figure 1. (G and H) RNA-scope analysis of Nmb (blue) and Trpm4 (red) transcript expression in the RTN (G) and the spinal trigeminal tract (H; sp5). The boxed regions are shown at higher power (G2 and H2) and with Trpm4 labeling overlaid on DAPI nuclei stain (G3 and H3). Scale bars, 50 μm. (I) Rostrocaudal distribution of Trpm4-expressing cells (mean ± SEM) within the population of Nmb-expressing RTN neurons (n =…

Figure 3.. Calcium-dependent TRPM4 currents contribute to subthreshold oscillations in RTN neurons (A) Top, Oscillation-waveform voltage command applied from Vhold = −40 mV. Bottom, Oscillation-waveform-evoked currents in cells recorded with a Cs+-based pipette solution under control conditions and in the presence of 200 μM Cd2+ were subtracted to obtain the Cd2+-sensitive current (purple). (B) I-V curves of oscillation-waveform-induced currents reveal inward Cd2+-sensitive current throughout the oscillation waveform. (C) Effects on peak oscillation-waveform-evoked currents of Cd2+ (p < 0.0001, n = 7) and TTA-P2 (1 uM, p = 0.025, n = 6), both by paired t test. Box and whiskers plots in all panels as in Figure 1. (D and E) I-V curves of oscillation-waveform-induced currents before and during exposure to 9-pt (30 μM) (D) or CBA (50 μM) (E). (F) Effects of TRPM4 blockers on peak oscillation-waveform-evoked currents (9-pt, p = 0.0004, n = 7; CBA, p = 0.0025, n = 5, by paired t test.). (G and H) The I-V curves of 9-pt-sensitive oscillation-waveform-evoked current were eliminated after blocking calcium currents with Cd2+ (200 μM; G1) or strongly inhibited by inhibiting L-type channels with…

Figure 4.. Voltage-activated K+ channels constrain the subthreshold oscillation in RTN neurons (A) Subthreshold oscillations (0.5 μM TTX, at −40 mV with DC injection), before and after TEA (10 mM). (B) Phase-plane plot of averaged oscillation in control (blue) and TEA (orange). (C), Integrated power and mean frequency of subthreshold oscillations before and after TEA (mean ± SEM, n = 7). RM ANOVA with Sidak’s post hoc: F1,6 = 35.11, p < 0.001 for TEA effect on power, ****p < 0.0001; F1,6 = 0.5135, p = 0.5006 for TEA effect on frequency. (D) Subthreshold oscillations (0.5 μM TTX, at −45 mV and −40 mV with DC injection) under control conditions and in the presence of the KCNQ channel inhibitor ML252 (10 μM). (E) Effect of ML252 on integrated power and mean frequency of subthreshold oscillations at the indicated membrane potentials (mean ± SEM, n = 9). RM ANOVA with Sidak’s post hoc: F1,8 = 17.08, p = 0.0033 for ML252 effect on power, *p < 0.05; F2,16 = 4.265, p = 0.0328, for frequency x membrane potential test; +, p = 0.0231. (F and G) I-V plots of oscillation-waveform-induced currents in cells recorded with a K+-based pipette solution reveal outward currents sensitive to TEA (F) and ML252…

Figure 5.. Acidosis-induced membrane potential depolarization activates TRPM4-dependent subthreshold oscillations in RTN neurons (A and B) Effect on subthreshold oscillations (in TTX, 0.5 μM) of repeated exposures to acidified bath under control conditions and then during bath application of vehicle (DMSO; A) or 9-pt (30 μM; B). The cells were initially set at −50 mV using DC injection, which remained fixed throughout the experiment. (C and E) Integrated power of membrane oscillations in RTN neurons under control conditions and then either in vehicle (C; n = 7) or in 9-pt (E; n = 6). RM ANOVA with Tukey’s post hoc test: for vehicle, F1,6 = 2.186, p = 0.19; for 9-pt, F1,5 = 14.4, p = 0.013; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Box and whiskers plots in all panels as in Figure 1. (D and F) Acidosis-evoked membrane depolarization under control conditions and then either in vehicle (D; p = 0.27; n = 7) or in 9-pt (F; p = 0.5; n = 6), by paired t test. (G and H) Representative rate histograms (top) and cell-attached recordings (bottom) of spontaneous firing responses of RTN neurons during changes of bath pH before and after exposure to vehicle (DMSO; G) or 9-pt (H). Recordings in…

Figure 6.. TRPM4 supports activation of RTN neurons by 5-HT (A–C) Firing rate histograms of RTN neuron firing rate (cell-attached recordings) under control conditions and in the presence of 5-HT (10 μM) before and after exposure to either vehicle (DMSO; A) or 9-pt (C). Recordings in the presence of synaptic blockers, but no TTX. (B and D) Firing frequency response to 5-HT under control conditions and after exposure to vehicle (C; n = 6) or 9-pt (D; n = 7). RM ANOVA with Tukey’s post hoc test: For vehicle, F1,5 = 2.57, p = 0.17; for 9-pt, F1,6 = 45.95, p = 0.0005; **p < 0.01, ****p < 0.0001. Box and whiskers plots as in Figure 1. (E) Representative traces of subthreshold oscillation before and during 5-HT (in TTX, at −40 mV with DC injection).

Figure 7.. TRPM4 inhibition in RTN reduces respiratory output in mice (A and C) Effect on diaphragm EMG (dEMG) of bilateral injections (30–40 nL) of vehicle and 9-pt (A; 30 μM) or CBA (C; 50 μM) in the RTN of freely breathing, anesthetized adult Phox2b::GFP mice. LHS, left hand side; RHS, right hand side. (B and D) Poincaré analysis of breath-to-breath frequency after vehicle, 9-pt (B), and CBA (D) from the corresponding experiments (in A and C). (E) Transverse brainstem section showing location of bilateral injection sites (marked by red fluorescent latex beads) in the RTN; expanded image shows the close proximity of the left injection site to GFP+ RTN neurons. ChAT, choline acetyltransferase. (F) Distribution of RTN injection sites of 9-pt (n = 6) and CBA (n = 10). (G) Grouped data (mean ± SEM) for on-target bilateral injections of vehicle, 9-pt, or CBA in the RTN. For fR and fR × dAmp, there was a significant interaction between injection number and drug for both 9-pt and CBA, relative to vehicle. 9-pt versus vehicle: fR, F2,39 = 30.43, p < 0.0001; fR × dAmp, F2,39 = 6.417, p = 0.0039. CBA versus vehicle: fR, F2,42 = 13.63, p < 0.0001; fR × dAmp, F2,42 = 3.299, p = 0.0467. **/##p < 0.01,…