Figures and data
Activation of the SP5C is correlated with tussive-evoked cough responses in mice.
(A) Diagram illustrating the setup for cough monitoring in awake mice. (B) Representative traces of box flow and audio signals after exposure to nebulized saline (left) or 1% NH3 (right) in WT mice. Triangles indicate cough responses. (C) Expanded traces of box flow and audio signals from (B) (highlighted by red dashed rectangle), showing eupnoea and a cough along with its corresponding audio signal. I-inspiration; C-compression; E-expiration. (D) Top, raster plot of coughs of individual mice exposed to nebulized NH3 (green bar). Bottom, the number of cough per minute. n = 10 mice. (E) Summary of the number of coughs evoked by various tussigenic stimuli. (F) Experimental strategy and representative images of TRAP2 labeling brainstem neurons during NH3 or saline nebulization. (G) NH3 exposure evoked strong cough-related neuronal Ca2+ signals in the SP5C as measured by in vivo fiber photometry. Individual trials of Ca2+ signals registered to the peak airflow (time = 0, dashed line) during cough. H the average Ca2+ signals in the SP5C of individual mice. n = 3.
Cough responses with variation in the boxflow waveforms can be observed across different tussive stimuli.
(A) Representative box flow and audio signals of two cough events evoked by nebulized CA (0.1M).
(B) Representative box flow and audio signals of two cough events evoked by nebulized capsacin (10 µM).
(C) Representative box flow and audio signals of two cough events evoked by nebulized NH3 (1%).
(D) Representative box flow and audio signals of two sneeze events evoked by nebulized capsacin (10 µM).
The neural activity in the NTS or VRG is correlated with tussive-evoked cough responses in mices.
(A) Representative images of TRAP2 labelled neurons in the VRG, PBN, PAG, and the NTS following NH3 exposure in TRAP2::Ai9 mice.
(B) Individual (left) and average (right) cough-correltaed GCaMP6s signals in the NTS recorded by in vivo fiber photometry.
(C) Individual (left) and average (right) cough-correltaed GCaMP6s signals in the VRG recorded by in vivo fiber photometry.
The SP5C is necessary for tussive-evoked cough responses in mice.
(A) Experimental strategy, combining stereotactic injection of AAV overexpressing Cre with transgenic mice of neurexin1/2/3 conditional knockout mice, to impair synaptic outputs of the SP5C. (B) Representative traces of box flow signals following exposure to 1% NH3 in mice with or without pan-neurexin deletion in the SP5C. (C) Summary of NH3-evoked cough responses in Ctrl and Nrxn123 TKO in the SP5C. (D-E) Representative traces of box flow signals following exposure to 1% NH3 in mice with EGFP or hM4Di expression in the SP5C when saline (D) or DCZ (E) was injected intraperitoneally. (F) Summary of NH3-evoked cough with and without chemogenetic inhibition of the SP5C.
Data are means ± SEM. Statistical difference was assessed by Student’s t-test. (***P < 0.001).
The VRG is necessary for tussive-evoked cough responses in mices.
(A)Strategy and fluorescence image showing AAV mediated pan-neurexin ablation in the VRG.
(C) Representative box flow signals of mice without or with pan-neurexin deletion in the VRG following NH3 exposure (left) and the summary of cough number (right).
Optogenetic stimulation of SP5C excitatory neurons is sufficient to trigger robust cough activities in mice.
(A) Strategy for optogenetic stimulation of specific cell types in the SP5C. (B-C) Representative box flow signals following optogenetic stimulation of VGlut2+ excitatory neurons, or VGAT+ inhibitory neurons, in the SP5C. (D) Experimental strategy of optogenetic stimulation of CaMKII+ excitatory neurons in the SP5C. (E) Representative box flow signals of mice during optogenetic stimulation of CaMKII+ neurons in the SP5C. (F) Summary of the number of coughs (converted into the rate in minute) evoked by optogenetic stimulation at various frequencies.
Data are means ± SEM. Statistical difference was assessed by Student’s t-test. (**P < 0.01; ***P < 0.001).
The SP5C projects to the VRG to trigger cough in mice.
(A) Schematic and time course of the retrograde tracing strategy. (B) RV labeling of VRG neurons receiving monosynaptic inputs directly from the SP5C. (C) Schematic of the anterograde tracing strategy. (D) AAV2/1 labeling of the SP5C afferent terminals innervating VRG neurons. (E) Strategy of optogenetic stimulation of SP5C projections to the VRG and simultaneous recording of Ca2+ signals from VRG neurons. (F) Representative box flow signals during optogenetic stimulation, at either high or low frequency, of the SP5C projections to the VRG. (G) Summary of the number of coughs evoked by optogenetic stimulation. (H) Summary of the GCaMP6s signals evoked by optogenetic stimulation of the SP5C projections to the VRG.
Data are means ± SEM. Statistical difference was assessed by Student’s t-test. (***P < 0.001).
The SP5C neurons project their terminals to the respiratory network, including the cVRG (A), rVRG (B) and preBotzinger complex (PrBo) (C).
Elevating SP5C neuronal excitability induces spontaneous cough and cough hypersensitivity in mice.
(A) Experimental strategy (left) and fluorescence image illustrating overexpression of NaChBac in the SP5C. (B-C) Representative traces of box flow and audio signals before and after exposure to NH3 in mice overexpressing EGFP as control (B) or NaChBac (C) in the SP5C. (D) Summary of the number of coughs per minute before, during and after NH3 exposure. (E) Summary of the cough latency (the time of first cough recorded after NH3 nebulization). (F) Representative traces of box flow signals before and after injection of DCZ in mice overexpressing NaChBac in the SP5C and HM4Di in the cVRG. (G) Summary of the number of spontaneous cough events before and after chemogenetic inhibition of the cVRG.
Data are means ± SEM. Statistical difference was assessed by Student’s t-test. (*P < 0.05; ***P < 0.001).
Functional properties of excitatory neurons in the SP5C.
(A) Representative action potenial (AP) traces of Sp5 neurons evoked by current injection at increasing intensity, showing the neuron’s capability for firing APs at high frequencies.
(B) Summary of AP firing frequency of SP5 neruons by injecting currents of increasing intensity.
(C) Summary of cell membrane resistance (left) and minimum currents required for evoking AP for SP5 neurons (right).
(D) Representative AP traces of Sp5 neurons stimulated by 1 Hz 470 nm light stimulation.
(E) Representative AP traces of Sp5 neurons stimulated by 20 Hz (left) and 50 Hz (right) 470 nm light pulses, indicating the reliability of neuronal firing in response to high-frequency optogenenetic stimulation.
NaChB-overexpressing neurons exhibit spontaneous action potentials and sustained depolarization to injected currents.
(A-B) Representative SP5 neuron overexpressing NaChBac fired spontaneous APs at high frequency (A) and evoked APs by injected current at the threshold level(B).
(C-D) Another SP5 neuron overexpressing NaChBac fired spontaneous APs but at a revlatively low frequency (C) and evoked APs by injected current at the threshold level (D).
Overexpression of NaChBac enhances neuronal activity in the SP5C following NH3 nebulization.
(A) Top, representative image showing TRAP2-labelled neurons after nebulized NH3 in TRAP2::Ai9 mice overexpressing NaChBac in the SP5C. Bottom, Enlarged region of interest highlighted by the blue rectangle in the Top image.
(B) Top, representative image showing TRAP2-labelled neurons after nebulized saline in TRAP2::Ai9 mice overexpressing NaChBac in the SP5C. Bottom, Enlarged region of interest highlighted by the blue rectangle in the Top image.
