Animals
All animal procedures followed ethical guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee at Harvard Medical School. Mice were maintained under constant environmental conditions (23 ± 1 °C, 46 ± 5% relative humidity) with food and water provided ad libitum under a 12 h–12 h light–dark cycle. All of the studies used adult male and female mice (aged 6–24 weeks) in comparable numbers from mixed genetic backgrounds. Tmie-, Gpr68- and Otop1-knockout mice were gifts from J. Holt, A. Patapoutian and E. Liman. All of the other mice were purchased from Jackson Laboratory, made in the laboratory and then deposited at Jackson Laboratory, or received as gifts and later deposited in the Jackson Laboratory: C57BL/6J (00664), Vglut2-ires-cre (16963), Npy2r-ires-cre (29285), Piezo2-ires-cre (27719), Phox2b-cre (16223), P2ry1-ires-cre (29284), Pvalb-t2a-cre (12358), Glp1r-ires-cre (29283), Oxtr-t2a-cre (31303), lsl-ChR2 (12569), lsl-DTR (07900), lsl-SALSA (31968), lsl-tdTomato (07914), loxP-Piezo2 (27720), loxP-Piezo1 (29213) and Snap25-GCamp6s (25111).
Physiological measurements
Arterial blood pressure was measured in anaesthetized mice (1.5–2% isoflurane in oxygen). The carotid artery or femoral artery was cannulated through a custom-built fluid catheter (Braintree Scientific RPT015, MRE033, MRE065) attached to a pressure transducer (BioPac, TSD104A; Sensor, RX104A) amplified by the Biopac DA100C system. Right atrial pressure was measured by inserting a catheter through the external jugular vein to access the atrium. Electrocardiograms were performed using needle electrodes placed subcutaneously on the right forepaw and left hindpaw, amplified using the Biopac ECG100C system. Breathing was measured using a pressure transducer (Harvard Apparatus, Differential Pressure Transducers MPX) within the isoflurane delivery device, amplified by the Biopac DA100C system. For some experiments, the jugular or femoral veins were cannulated for blood withdrawal or saline (0.9% NaCl) infusion manually or with a syringe pump (NE-1000, New Era Pump Systems). Data were acquired using the Biopac MP160 system with the Acknowledge software.
Tilt-table test
Anaesthetized mice (1.5–2% isoflurane in oxygen) were immobilized on a surgical platform with adhesive, and prepped for measurements of blood pressure, heart rate and breathing. The isoflurane concentration was then reduced to 1.2–1.5% until the breathing rate stabilized at around 60 breaths per minute. The blood pressure transducer was kept level with the catheter site to avoid a hydrostatic effect during rotation. The surgical platform with the mouse was magnetically secured to a tilt table, which was connected to an accelerometer for movement recording. The mouse was manually tilted to different positions, remaining in each position for at least 1 min while the physiological parameters were recorded (Biopac).
Haemorrhage model
Mice were anaesthetized (1.5–2% isoflurane in oxygen) and placed onto a heating pad to maintain the body temperature above 35°C. Heparin (200 U kg−1 in PBS) was administered through the right external jugular vein or tail vein and isoflurane was lowered (1.2–1.5%) until the breathing rate stabilized at around 60 breaths per minute. After 10 min, the tail was transected (2 cm from the tip) with a scalpel to induce bleeding. Blood was collected in an Eppendorf tube and weighed at 30 min and at the survival end point. Survival was continuously assessed by detectable breathing. Animals of different genotypes were used in a random order to which the experimenter was blinded for analyses of survival and heart rate.
Neuron ablation
Vagal sensory neurons were ablated as previously described by serial injection (10–20 injections of 10 nl) of DT (Sigma-Aldrich, D0564) solution (5–20 μg ml−1 DT, 0.05% Fast Green FCF Dye with PBS) into surgically exposed nodose/jugular/petrosal (NJP) superganglia with a Nanoject III injector (Drummond). Control mice were injected with PBS lacking DT. Mice were allowed to recover for at least 3 weeks after injection, and nodose ganglia were collected and immunostained for DTR after experiments to determine the extent of ablation.
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