R ES E A RC H

NEUROSCIENCE transduction is not clear. PIEZO1 and PIEZO2

are mechanically activated ion channels that play
crucial roles in several mechanotransduction pro-
PIEZOs mediate neuronal cesses (10). PIEZO1 is prominently expressed
in the cardiovascular system (11, 12), and PIEZO2

sensing of blood pressure and is abundant in various populations of sensory

neurons (13–15).
We assessed Piezo1 and Piezo2 transcript ex-
the baroreceptor reflex pression in nodose and petrosal ganglia, where
baroreceptor cell bodies are located (1). These
Wei-Zheng Zeng1, Kara L. Marshall1, Soohong Min2, Ihab Daou1, Mark W. Chapleau3,4,
ganglia are fused with each other and with the
jugular ganglion in mice. Piezo1 and Piezo2 were
Francois M. Abboud3, Stephen D. Liberles2, Ardem Patapoutian1*
highly expressed in the nodose-petrosal-jugular
ganglion complex (NPJc) (Fig. 1A). Similar num-
Activation of stretch-sensitive baroreceptor neurons exerts acute control over
bers of cells were identified that highly expressed
heart rate and blood pressure. Although this homeostatic baroreflex has been
either Piezo1 or Piezo2 exclusively (123 and 124
described for more than 80 years, the molecular identity of baroreceptor
cells with each transcript, respectively, Fig. 1B).
mechanosensitivity remains unknown. We discovered that mechanically activated
A small population of neurons expressed both
ion channels PIEZO1 and PIEZO2 are together required for baroreception. Genetic
(43 double Piezo-positive cells, or 14.8% of all
ablation of both Piezo1 and Piezo2 in the nodose and petrosal sensory ganglia
Piezo-expressing cells, n = 6 mice, Fig. 1B).
of mice abolished drug-induced baroreflex and aortic depressor nerve activity. Awake,
To test whether Piezo1 and Piezo2 are ex-
behaving animals that lack Piezos had labile hypertension and increased blood

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pressed in baroreceptors, we performed retro-
pressure variability, consistent with phenotypes in baroreceptor-denervated animals
grade labeling of carotid sensory neurons. We
and humans with baroreflex failure. Optogenetic activation of Piezo2-positive
injected fluorescent cholera toxin B (CTB) (16)
sensory afferents was sufficient to initiate baroreflex in mice. These findings
into the carotid sinus beneath the serosal vessel
suggest that PIEZO1 and PIEZO2 are the long-sought baroreceptor mechanosensors
covering. All CTB-positive neurons detected in
critical for acute blood pressure control.
the NPJc from eight mice were quantified for

B
lood pressure (BP) is tightly regulated to
ensure that the body is prepared to meet
varied daily activity demands. Mechanisms
that change blood volume control long-
term BP regulation. Within seconds and
minutes, BP regulation is initiated primarily by
baroreceptors, a class of stretch-sensitive neurons
within the nodose and petrosal ganglia with
peripheral projections in the walls of the aorta
and carotid sinus (1, 2). An increase in BP stretches
baroreceptor nerve endings to trigger afferent
signals that are transmitted to the central nervous
system. The consequences of baroreceptor acti-
vation are a decrease in heart rate (HR), cardiac
output, and vascular resistance that counteract
the initial increase in BP (1, 2). Compromised
baroreceptor function predicts arrhythmias and
premature death in humans with postmyocardial
infarction and heart failure (3, 4).
Several ion channels (5–9) have been sug-
gested to contribute to baroreception. How-
ever, substantial residual baroreflex is observed
when these channels are ablated, implicating
the involvement of other sensory systems. None
of the candidate ion channels have been directly
activated by mechanical stimuli in heterologous
systems, which may lack accessory tethering
molecules to form a mechanosensory complex.
Furthermore, whether these channels are acting
as sensors or play a role downstream of mechano-
the presence of Piezo1 or Piezo2 transcript (Fig. 1,
C to F). Six out of 95 retrogradely labeled cells
were Piezo1-positive, and eight were Piezo2-
positive (Fig. 1B). Piezo-negative cells were likely
chemoreceptors, which abundantly innervate
the carotid sinus but do not require mecha-
nosensitivity. None of the 95 CTB-labeled cells
1
Howard Hughes Medical Institute, Neuroscience Department,
Dorris Neuroscience Center, The Scripps Research Institute, La
Jolla, CA 92037, USA. 2Howard Hughes Medical Institute,
Department of Cell Biology, Harvard Medical School, Boston,
MA 02115, USA. 3Abboud Cardiovascular Research Center,
Department of Internal Medicine and Molecular Physiology
and Biophysics, Carver College of Medicine, University of
Iowa, Iowa City, IA 52242, USA. 4Veterans Affairs Medical
Center, Iowa City, IA 52242, USA.
*Corresponding author. Email: ardem@scripps.edu

Fig. 1. Expression of Piezo1 and Piezo2 transcripts in NPJc. (A) Z-projection
of NPJc tissue after fluorescent in situ hybridization with probes
targeting Piezo1 (red) and Piezo2 (cyan). Nuclei are labeled with 4′,6-
diamidino-2-phenylindole (DAPI, blue). Arrows mark double Piezo-positive
cells. (B) Quantification of transcript labeling area as a fraction of total
cell area (n = 290 cells, six mice). Each dot represents one cell. Numbers
in parentheses indicate number of cells. P1, Piezo1; P2, Piezo2. (C to F) NPJc
cell bodies back-labeled by carotid sinus CTB injections [(C) and (D),
green] and Piezo transcript [(E) and (F), red and cyan]. Arrows indicate
a Piezo2-positive cell in (C) and (E) and a Piezo1-positive cell in (D)
and (F). (G) Quantification of Piezo transcript labeling area in CTB-positive
cells (n = 95 cells, eight mice). Piezo-negative cells are not shown.

Zeng et al., Science 362, 464–467 (2018) 26 October 2018 1 of 4

R ES E A RC H | R E PO R T

were double Piezo-positive. These data suggest
that a subset of neurons that innervate the
carotid sinus (which include mechanoreceptors
and chemoreceptors) express either Piezo1 or
Piezo2 (Fig. 1G). We hypothesized that these
cells could function as baroreceptors.
We therefore crossed Piezo floxed mice to the
Phox2bCre line, which express Cre recombinase
in epibranchial placode-derived ganglia (e.g.,
nodose and petrosal) but not in neural crest–
derived ganglia (jugular, trigeminal, and dorsal
root) (17). We first analyzed the baroreflex in
anesthetized mice in response to phenylephrine
(PE). PE induces rapid vasoconstriction (6), which
elevates BP. Increased BP then triggers barore-
ceptor activity and induces a reflex decrease in
HR. PE-induced baroreflex changes were com-
pared in conditional double-knockout mice (dKO;
Phox2bCre+;Piezo1 f/fPiezo2f/f) and Cre-negative
wild-type littermates (WT). Infusion of PE into
the jugular vein produced a dose-dependent and
transient increase in systolic BP and a conse-
quent decrease in HR, reflecting baroreflex con-
trol (6) (Fig. 2A). The PE-induced HR reduction
[−29 ± 20 versus −234 ± 24 beats per minute
(bpm), P < 0.001] and decreased baroreflex sen-
sitivity (−0.6 ± 0.4 versus −5.0 ± 0.5 Dbpm/
DmmHg, P < 0.001) were essentially abolished
in the dKO mice (Fig. 2, A to D). PE-induced sys-
tolic BP increase in dKO mice was significantly
higher than in WT littermates (55.7 ± 3 versus
45.7 ± 6 mmHg, P <0.05) (Fig. 2, A and B). HR
response to sodium nitroprusside–induced acute
baroreceptor unloading was also absent in dKO
mice (fig. S1, A to C). By contrast, Phox2bCre+;
Piezo1f/f (P1cKO) and Phox2bCre+;Piezo2f/f (P2cKO)
single-knockout mice showed no difference in
PE-induced change of baroreflex compared with
WT littermates (Fig. 2, B to D). We focused re-
maining analyses primarily on dKO mice.
We next measured aortic depressor nerve (ADN)
activity during a rise of BP induced by PE. We

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Fig. 2. Baroreflex is abolished in nodose and petrosal ganglia–specific
dKO mice. (A) Cardiovascular recordings show PE-induced baroreflex in
WT mice but no baroreflex in dKO littermates. BP, raw blood pressure
signal. SYS, systolic blood pressure derived from raw BP. (B to D) Changes
in systolic BP (B), HR (C), and baroreflex (D) (10 s after intravenous
injection of PE) in knockout (KO) mice. Number of animals shown
in bars (B) also apply for (C) and (D). Piezo1 KO indicates P1cKO mice;
Piezo2 KO indicates P2cKO mice; Piezo1 Piezo2 KO indicates dKO mice.
All WT are littermates. (E) Traces show BP and ADN activity induced by
PE and sodium nitroprusside injection in a WT and a dKO mouse. SNP,
sodium nitroprusside. (F) Statistical analysis of drug-induced ADN activity
in WT (n = 16) and dKO (n = 11) mice. (G) Raw BP and ADN activity
example before and after PE injection. Expanded time scale showed bursts
of ADN activity in phase with individual arterial pulses in WT. No integrated
activity is observed in dKO mice. *P < 0.05, ***P < 0.001, and n.s. is statistically
not significant by unpaired Student’s t test; data are means ± SEM.

Zeng et al., Science 362, 464–467 (2018) 26 October 2018 2 of 4

R ES E A RC H | R E PO R T

observed a lack of drug-induced ADN activity in

dKO mice compared to WT mice (−131.9 ± 163.9
versus 5558 ± 1234 normalized area under curve
of integrated ADN activity, P < 0.001; Fig. 2, E to
G). The dKO mice had no appreciable responses
during both phasic and tonic phases of PE-
induced ADN activity (fig. S1, D and E). This is
not due to gross anatomical deficits, because we
observed comparable baroreceptor ending den-
sities within the aortic arch of dKO and WT mice
(fig. S2).
Impaired baroreceptor function leads to dys-
regulation of BP, including volatile hypertension
and increased BP variability in humans (18–20).
We examined daily BP variability in freely mov-
ing, conscious mice using a telemetric sensor (6).
Fig. 3. Increased BP variability in conscious nodose and petrosal ganglia–specific dKO mice. The dKO mice showed significantly increased
(A) Continuous measurements of MAP and HR over 72 hours, binned by hour. The differences between mean arterial pressure (MAP) during their ac-
groups were significant during the night (gray shading, two-way analysis of variance, data are means ± tive time (gray shading, 6 p.m. to 6 a.m.) com-
SEM). (B) Average MAP and HR during the day (6 a.m. to 6 p.m.) and night (6 p.m. to 6 a.m.). pared with WT littermates (112 ± 0.4 versus 95 ±
(C) sBRS, expressed as change in PI (ms) per change in systolic BP (mmHg), was significantly reduced 0.5 mmHg, P < 0.001) (Fig. 3, A and B, and figs.

Downloaded from http://science.sciencemag.org/ on October 29, 2018

in dKO mice (n = 17 mice) compared with WT mice (n = 15 mice). (D) Frequency distribution histogram S3, A and B, and S4). The HR of dKO mice was
of the systolic BP over 72 hours. Red arrows indicate wider distribution of BP in dKO mice. (E) BP slightly increased during active times compared
variability reported as standard deviation from the 72-hour period. (F) Maximum and minimum BP values. with that of WT mice (583 ± 3 versus 566 ±
P values are indicated in the bars. *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. is statistically not significant. 3 bpm, P < 0.001), whereas the HR remained
Unpaired Student’s t test was used unless indicated otherwise; data are means ± SEM. n = 7 to 17 mice. unchanged during inactive times (6 a.m. to 6 p.m.,
532 ± 3 versus 536 ± 3 bpm, not significant)
(Fig. 3B). No difference in locomotor activity
was observed between dKO and WT mice (fig.
S3C), ruling out the possibility that activity
caused the increased BP and HR in dKO mice.
We scanned telemetry data for spontaneous
changes in systolic BP and pulse interval (PI)
consistent with a baroreflex relationship. This
method (sequence technique) is used to nonin-
vasively assess baroreflex function (21, 22). The
spontaneous baroreflex sensitivity (sBRS) is de-
fined as the slope of changes in systolic BP versus
PI from 1 hour of recording. sBRS was severely
reduced in dKO mice (2.0 ± 0.1 versus 3.8 ±
0.2 ms/mmHg for WT, P < 0.001, Fig. 3C). Sinoaortic
baroreceptor denervated mice also show residual
sBRS (22), and this may be due to compensation
from other sensory systems.
We compared the BP variability of WT and
dKO mice. The systolic BP values of dKO mice
were distributed in a broader range than those of
WT littermates (Fig. 3D). Variability was greatly
enhanced in dKO mice (7.9 ± 0.3 versus 6.1 ±
0.3 mmHg in WT, P < 0.001, Fig. 3E). We quan-
tified the range of BP variability of mean, sys-
tolic, and diastolic BP within each group in a
72-hour period. Maximum values of BP from
dKO mice were significantly higher than those
from WT littermates, whereas minimum values
were significantly lower (Fig. 3F). Lastly, homo-
vanillic acid concentrations in dKO mouse urine
Fig. 4. Piezo2-positive sensory neurons acutely control blood pressure. (A) Schematic were significantly higher than those in WT urine
depiction of the optogenetic strategy. The carotid sinus and vagus nerves were illuminated to activate (13.9 ± 0.05 versus 12.1 ± 0.06 mg/ml, fig. S3D),
ChR2-expressing Piezo2-sensory neurons. The optical fiber was placed on area 1, vagus nerve trunk; suggesting an increase in hormone norepineph-
area 2, superior laryngeal branch; and area 3, carotid sinus. (B) Traces of cardiovascular effects rine concentration, as in human baroreflex
after focal vagus nerve illumination (light blue shading) in anesthetized Piezo2Cre−;ChR2-eYFP failure patients (18). There were no significant
(WT, black trace) and Piezo2Cre+;ChR2-eYFP mice (Piezo2Cre+, gray, blue, and pink traces). BP variability and sBRS differences in P1cKO
Numbers on the left (1 to 3) correspond to areas in (A). Blood pressure was measured by a pressure (fig. S5) and P2cKO (fig. S6) single-knockout
transducer cannulated in the left carotid artery. BP, carotid arterial pressure. (C) Light-induced mice compared with WT littermates. P2cKO mice
changes in BP and HR were calculated over the 10 s (n = 7 to 18 mice, as indicated; ***P < 0.001, showed a subtle hypotensive BP distribution
and n.s. is statistically not significant by unpaired Student’s t test; data are means ± SEM). (fig. S6).

Zeng et al., Science 362, 464–467 (2018) 26 October 2018 3 of 4

R ES E A RC H | R E PO R T
We next investigated whether stimulating
Piezo2-positive neurons can induce the baroreflex
in adult mice. We crossed Piezo2GFP-IRES-Cre
(Piezo2Cre) knockin mice with Cre-dependent
channelrhodopsin-2 (ChR2) reporter mice to
generate Piezo2Cre+;ChR2-eYFP mice (13) and
recorded the cardiovascular response to activat-
ing different regions of Piezo2-positive vagal
sensory nerves by optogenetics (Fig. 4A; eYFP,
enhanced yellow fluorescent protein gene). We
did not observe cardiovascular changes during
long optogenetic stimulation (5-ms pulses, 50 Hz,
10 s) of the vagal nerve trunk (area 1 in Fig. 4A; BP,
−5.3 ± 1.0%, and HR, −2.0 ± 1.0%; not significant)
(Fig. 4, A to C). Next, we focused on specifically
activating baroreceptor afferents. For aortic baro-
receptors, we stimulated the superior laryngeal
nerve branch, which carries afferent inputs from
the ADN (area 2 in Fig. 4A). For carotid baro-
receptors, we exposed the carotid sinus region
and directly stimulated the local nerve terminals
(area 3 in Fig. 4A). Light stimulations at both
locations caused an immediate decrease in both
BP and HR (area 2: BP, −55.6 ± 2.0%, and HR,
−50.5 ± 2.0%; area 3: BP, −37.5 ± 3.5%, and HR,
−32.3 ± 3.7%; P <0.001) compared with the un-
stimulated baseline (Fig. 4, B and C). A promi-
nent consequence of baroreceptor activation is
rapid inhibition of efferent sympathetic activ-
ity (1). We found that light-induced decrease in
HR was markedly attenuated after administra-
tion of the b-adrenergic receptor–blocker pro-
pranolol, indicating that the reflex bradycardia
was mediated primarily by inhibition of cardiac
sympathetic nerve activity (fig. S7). Piezo2Cre−;
ChR2-eYFP mice (WT) did not show any changes
during optogenetic stimulation in all three re-
gions (BP, −0.6 ± 0.6%, and HR, 0.4 ± 0.8%; not
significant; Fig. 4, B and C).
This study demonstrates that the mechani-
cally activated ion channels PIEZO1 and PIEZO2
are together required for arterial baroreceptor activ-
Zeng et al., Science 362, 464–467 (2018) 26 October 2018
ity and function. Baroreflex is critical to maintain
short-term BP homeostasis in mammals. The
long-term changes observed in HR and BP that
accompany baroreflex failure are complex. Acute
elimination of baroreceptor function (e.g., sino-
aortic denervation) causes immediate, large in-
creases in BP and HR (23, 24). Over time, the
mean BP decreases but remains labile hyper-
tensive, and BP variability is markedly increased
and persists (18–20, 24, 25). We observed a sig-
nificant increase in MAP during the active period
of the Piezo dKO mice that falls just under the
designation for hypertension (26), and dKO
mice also developed increased blood pressure
variability. These data show that losing PIEZO1
and PIEZO2 function recapitulates the pheno-
type observed in animal models (24, 25) and
humans with baroreflex failure (18–20). However,
we cannot exclude the possibility that sensory
mechanisms beyond the baroreceptors within
the vagus contribute to the observed increased
blood pressure.
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AC KNOWLED GME NTS
We thank D. Morgan, S. Ma, and K. Nonomura for assistance and
D. Ginty for the suggestion to assess the role of PIEZO2 in
baroreceptors. Funding: This work was supported by NIH
grants R01 DE022358 and R35 NS105067 to A.P. W.-Z.Z. was
supported by a postdoctoral fellowship from the George
Hewitt Foundation for Medical Research. S.D.L. was supported
Downloaded from http://science.sciencemag.org/ on October 29, 2018
by NIH grants DP1 AT009497 and OT2 OD023848. M.W.C
and F.M.A were supported by NIH grant P01 HL14388. A.P.
and S.D.L. are investigators of the Howard Hughes Medical
Institute. Author contributions: W.-Z.Z., K.L.M., and A.P.
designed experiments and wrote the paper. K.L.M. performed
in situ hybridization and baroreceptor innervation analysis.
W.-Z.Z. performed drug-induced baroreflex assessment,
telemetry sensor implantation, BP variability, and sBRS analysis.
W.-Z.Z. and I.D. performed optogenetics experiments. S.M.
performed ADN activity recordings in the S.D.L. laboratory.
M.W.C and F.M.A advised and trained W.-Z.Z., contributed
to technical approaches, and edited the manuscript.
Competing interests: The authors declare no competing
interests. Data and materials availability: All data
are available in the main text or the supplementary
materials.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/362/6413/464/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S7
References (27, 28)
29 June 2018; accepted 7 September 2018
10.1126/science.aau6324
4 of 4
PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex
Wei-Zheng Zeng, Kara L. Marshall, Soohong Min, Ihab Daou, Mark W. Chapleau, Francois M. Abboud, Stephen D. Liberles
and Ardem Patapoutian

Science 362 (6413), 464-467.

DOI: 10.1126/science.aau6324

Heart rate and blood pressure control

PIEZO1 and PIEZO2 are two mechanically activated ion channels that are highly expressed in lungs, bladder, and
skin. Zeng et al. found that both ion channels are expressed in sensory neurons of a ganglion complex that contribute to
the baroreflex, a homeostatic mechanism that helps to keep blood pressure stable (see the Perspective by Ehmke).

Downloaded from http://science.sciencemag.org/ on October 29, 2018

Conditional double knockout of PIEZO1 and PIEZO2 in these neurons abolished the baroreflex and disrupted blood
pressure regulation and heart rates in mice. These changes were very similar to those seen in patients with baroreflex
failure. In mice, selective activation of PIEZO2-expressing ganglion neurons triggered immediate increases in heart rate
and blood pressure.
Science, this issue p. 464; see also p. 398

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REFERENCES This article cites 28 articles, 5 of which you can access for free
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Supplementary Materials for

PIEZOs mediate neuronal sensing of blood pressure and the
baroreceptor reflex

Wei-Zheng Zeng, Kara L. Marshall, Soohong Min, Ihab Daou, Mark W. Chapleau,
Francois M. Abboud, Stephen D. Liberles, Ardem Patapoutian*
*Corresponding author. Email: ardem@scripps.edu

Published 26 October 2018, Science 362, 464 (2018)

DOI: 10.1126/science.aau6324

This PDF file includes:

Materials and Methods

Supplementary Text
Figs. S1 to S7
References
Materials and Methods
Mouse lines
All animal procedures were approved by the Institutional Animal Care and Use Committees of
The Scripps Research Institute in compliance with regulatory standards established by the
Association for Assessment and Accreditation of Laboratory Animal Care International
(AAALAC). The Piezo2GFP-IRES-Cre (Piezo2Cre) mouse line has been previously described
(13). Phox2bCre;Piezo1fl/fl mice were generated by breeding Phox2bCre mice (Jackson
Laboratories: #16223) with Piezo1fl/fl mice. Phox2bCre;Piezo2fl/fl mice were generated by
breeding Phox2bCre line with Piezo2fl/fl mice (13). Phox2bCre; Piezo1fl/flPiezo2fl/fl mice were
generated by breeding Phox2bCre line with Piezo1fl/flPiezo2fl/fl mice. Phox2bCre mice were also
crossed to Ai9 reporter mice (Jackson Laboratories: #7909) to specifically label baroreceptor
innervations. Piezo2Cre;ChR2 mice were generated by breeding Piezo2Cre mice with Ai32
(RCL-ChR2(H134R)/EYFP) mice (Jackson Laboratories: #12569).

Retrograde labeling of neurons innervating carotid baroreceptors

Surgery was carried out on 8 to 10-week-old C57BL/6J mice. Body temperature was
maintained at 37°C using a heating pad. After mice were anaesthetized by 1.5% isoflurane
supplied together with pure oxygen, the carotid sinus was exposed by a midline cervical incision.
A small volume (approximately 0.5 µL) of the neuronal fluorescent tracer Cholera Toxin Subunit
B Alexa Fluor 488 (Thermo Fischer Scientific, # C-34775) diluted to 2 mg/mL in phosphate
buffered saline was injected bilaterally into the carotid sinuses using a microsyringe (Hamilton).
Care was taken to inject underneath the serosal layer to enable dye access to the adventitial
surface. We targeted this area for its accessibility in the neck as compared to the aortic arch,
which sits within the chest cavity. The injection area was then dried with sterile cotton swabs to
minimize leaking. The animals were sutured and recovered for 4-6 days to allow for sufficient
retrograde labeling of cell bodies before ganglia were dissected.
To validate that CTB labeled neurons were originating from the carotid sinus and not
indiscriminately absorbed by nearby vagal axons, we performed a control experiment to test
whether dye leaking to surrounding areas would label nearby vagal afferents. CTB-Alexa 488
was injected into the carotid sinus as described above, and during the same surgery, the vagus
nerve trunk near the carotid sinus was isolated from surrounding tissue, and CTB-Alexa 647 was
applied directly to the nerve. After 4 days, we assessed nodose-petrosal-jugular ganglion
complex (NPJc) sections for the presence of both fluorophores. In six ganglia from three
bilaterally injected animals, we observed 25 cells positive for CTB-488, but only one CTB-647-
-positive cell. CTB enters neurons through their terminal endings, severed axons, or less robustly
via direct intra-nerve injection (27, 28). Therefore, dye leaking from the carotid sinus area into
nearby nerve trunks is unlikely to account for the vast majority of retrogradely labeled neurons
observed in these experiments. We conclude that carotid sinus injections are primarily labeling
carotid chemo- and baroreceptors.

In situ hybridization and analysis

The NPJc was isolated from adult mice, fresh frozen in liquid nitrogen, and cryosectioned at
20 µm. Fluorescent in situ hybridization was performed according to manufacturer’s instructions
(ACDBio: #323100) using probes against Piezo1 (ACDBio: # 400181) and Piezo2 (ACDBio:
#439971-C2).

2
 Stained sections were imaged with a Nikon C2 laser scanning confocal microscope (20×, NA
0.9) and z-stacks were acquired to capture the full thickness of the tissue section. Images were
analyzed using the Nikon Elements Analysis software. Regions of interest (ROI) were drawn
around single ganglion cell bodies using DAPI fluorescence as an indicator of a full cell within
the section. Cells bodies without DAPI staining in the nucleus were not analyzed to avoid double
counting. A threshold that eliminated the lowest 10% of intensity values was set to exclude
background staining, and the remaining pixel area was recorded within the ROI for each channel.
Area of transcript labeling was quantified as a fraction of total ROI area. For a given
CTB-positive cell, only the optical sections that contained that cell were made into a z-projection
before analysis. This minimized, but did not perfectly eliminate, cell overlap and false-positive
transcript labeling within an ROI. We set the cutoff for Piezo1 and Piezo2 transcript expression
as the median for the entire population of cells analyzed. Thus, to qualify as Piezo-positive, a cell
had to exceed the median fraction of transcript area labeling (Piezo1 = 0.064, Piezo2 = 0.086).
This served to exclude cells that had minimal transcript or had partial overlap of labeling from
another cell, but it is possible that the cells with little transcript also contain functional protein.

Drug-induced baroreflex
Mice were anaesthetized by 1.5% isoflurane and body temperature was maintained at 37°C
using a heating pad. Blood pressure was continuously measured by a pressure transducer (Biopac
TSD104A) cannulated to left carotid artery. Heart rate was measured by ECG, which was
recorded with three needle electrodes placed subcutaneously on the lower left chest (+), upper
right chest (-) and left hindpaw (ground), respectively and amplified with a differential amplifier
(MP150 data acquisition system, Biopac). The right jugular vein was cannulated with
micro-renathane tubing (MRE 040) for intravenous injection. Baroreceptor sensitivity was
evaluated by calculating heart rate during changes in systolic blood pressure 10 seconds after
intravenous injection of phenylephrine (PE) or sodium nitroprusside (SNP) (0.1 mg/mL, 50 µL).

Measurements of Aortic Depressor Nerve (ADN) activity

The mice were anesthetized with urethane (1 mg/g, i.p.) with supplemental doses as deemed
necessary. The trachea was cannulated using a PE-50 tubing for ventilation and the right carotid
artery was cannulated to a pressure transducer (Biopac TSD104A) for BP monitoring. The left
cervical region was exposed and the landmark for locating the ADN is the characteristic cluster
of dense fat tissue surrounding the region, where ADN joins the superior laryngeal nerve. ADN
trunk was placed on miniaturized bipolar platinum electrodes as previously described (6). To
specifically record the sensory component of ADN activity, the nerve fiber after the electrode
hook was crushed. The nerve activity was amplified using a Grass P511 AC amplifier (Natus
neurology) and integrated using a signal integrator (Elenco, RS-400). The neurogram was
recorded using the AcqKnowledge5 program.
Aortic baroreceptor sensitivity was evaluated by calculating ADN activity during changes in
blood pressure induced by intravenous injection of 50 µL phenylephrine (PE, 2 mg/mL in saline)
and 50 µL sodium nitroprusside (SNP, 1.25 mg/mL in saline) into the left femoral vein. For
quantification, Matlab program was used to calculate the area under curves from integrated ADN
activities. Normalized values of area under curve are generated by using drug-induced ADN
activity (from PE injection to 2-minute after SNP injection) subtracted by the steady state ADN
activity (2-minute before PE injection). The phasic ADN activity was analyzed within 30

3
seconds following PE injection. Tonic ADN activity was analyzed over 30 seconds when BP
reached a plateau (Fig. S1 D).

Telemetry recording of BP, HR and pulse interval in conscious mice and analysis of spontaneous
baroreflex sensitivity
The continuous recording of blood pressure and heart rate by telemetry in conscious rodents
has been established. Anesthesia of mice was induced and maintained with 3% and 1.5%
isoflurane with pure oxygen, respectively. Under anesthesia, the blood pressure probe (HD-X11,
DSI, USA) was inserted into the left carotid artery caudal to the carotid bifurcation and advanced
to place the pressure-sensing catheter tip in the aorta. The body of the probe was placed in a
subcutaneous pocket created in the right flank. The wound was closed and sutured, and body
temperature was maintained at 37°C using a heating pad until sternal recumbency had been
recovered. At least 14 days after recovery, diurnal changes in blood pressure, heart rate, and
locomotor activity were measured in the conscious freely moving state, over a minimum of
24-hour at a sampling rate of 1,024 Hz every 5 second (Ponemah v5.20, DSI, USA). Average
mean arterial blood pressure and heart rate in 1-hour time intervals were plotted continuously for
72 hours from 10:00 am to 10:00 am. The frequency distribution histogram of the systolic blood
pressure in a 72-hour period was sampled in 1 min time intervals and plotted with the bandwidth
of 10 mmHg pressure range. The highest and lowest 2.5% of the recorded blood pressure values
were discarded to remove possible noise. Representative telemetry traces for 24-hour recording
of mean blood pressure are visualized by Ponemah v5.20.
The spontaneous baroreflex sensitivity was estimated from reciprocal fluctuations in blood
pressure and heart rate by the sequence method (6) and analyzed using HemoLab software (Ver.
20.5, Harald Stauss Scientific). Recordings were obtained at sampling rate of 1,024 Hz over a
period of 60 min. The number of pulse sequences where changes in systolic blood pressure and
pulse interval (PI) correlated positively (r2 > 0.85) for four consecutive beats or more were
counted. The number of all baroreflex sequences per mouse is 98.5 ± 11.3 within one-hour
recording, which enable us to obtain an accurate estimation of the mean baroreflex slope of each
mouse. The average ratio of the systolic BP-PI sequences (∆ms/∆mmHg) provided a measure of
the baroreceptor reflex sensitivity (6).

Optogenetic stimulation of the vagus nerve and cardiovascular measurements

Animals were anesthetized by 1.5% isoflurane supplied together with pure oxygen and
maintained at normal body temperature. The left carotid sinus was surgically exposed and an
optic fiber (200 mm core, NA=0.66, Prizmatix) coupled to a diode laser light source (462 nm,
300 mW, Shanghai Laser & Optics Century) positioned for focal illumination beneath the 1)
vagus trunk, 2) superior laryngeal branches and 3) carotid sinus (Fig. 4A). To activate aortic
baroreceptors, we stimulated the superior laryngeal nerve branch (area2). This area was targeted
because accessing the aortic arch and aortic depressor nerve within the chest and neck cavity is
challenging. Light stimulation (5 ms pulses, 4.5 mW intensity, 50 Hz, 10 s) was controlled by a
pulse train generator (Prizmatix). Cardiovascular parameters were measured using an amplifier
coupled pressure transducer (Biopac TSD104A) cannulated into the left carotid artery. Heart rate
was simultaneously derived from the carotid arterial pressure pulse with an amplifier (500 Hz
sampling rate, MP150 data acquisition system, Biopac). Data analyzed in 5-second bins (Fig.
4B) were normalized by comparison to values obtained during a 30-second baseline period.

4
Statistics
All statistical analysis of data is detailed in the figure legends. All statistical analyses were
performed with GraphPad Prism 6. When unpaired Student’s t-test was applied, similarity of
variance between groups was confirmed by F test. All statistical analyses were two-sided.

Sample size choice

The specific number of independent experiments or animal numbers used for all experiments
is outlined in the corresponding figures or their legends. All mice tested were with littermate
controls. All experimental procedures were performed in a blinded manner. No randomization
was applied. No statistical method was used to predetermine sample size, but these were chosen
to be comparable to previous studies of the mammalian cardiovascular system.

Supplementary Text
SNP-induced unloading of baroreflex and PE-induced phasic/tonic ADN activity in dKO and WT
mice
We assessed the acute baroreceptor unloading by intravenous injection of sodium nitroprusside
(SNP, 0.5 mg/kg), which induces rapid vasodilation and reduces BP. We observed a comparable
SNP-induced decrease of BP between dKO and WT littermates (dKO -34.0 ± 1.0%, WT -38.8 ±
3.7%, not significant, by Student’s t-test, Fig. S1 A, C). SNP-induced HR increase (-2.8 ± 2.1%
versus 14.5 ± 1.9%, p<0.001, by Student’s t-test, Fig. S1 A, B) were severely attenuated in the
dKO mice compared to WT littermates. In addition, ADN activity during SNP injection is
abolished in dKO mice (Fig. 2E). These results, together with PE-induced baroreceptor activation,
suggest that the baroreflex sensitivity is abolished in dKO mice for both directions.
We did observe rapid rises of BP and ADN activities in most of our recordings by intravenous
injection of drugs through femoral veins. There is a distribution of time delays between PE
injection and BP rise, and it so happens that the one chosen for the original figure happened to have
a bit of a longer delay. We re-analyzed 7 mice (3 WT, and 4 dKO) in which we observed a rapid
rise of BP within 30 seconds. The dKO mice had essentially no appreciable responses during both
the phasic and tonic phases (Fig. S1 D-E)

Sympathoinhibition during Piezo2 baroreceptor activation

We assessed the sympathoinhibition during Piezo2 optogenetic activation before and after
intravenous injection of propranolol (5 mg/kg), a nonselective β-adrenoreceptor antagonist that
blocks HR responses to changes in sympathetic nerve activity. As expected, administration of
propranolol significantly reduced the heart rate (-24.1 ± 1.6%, p＜0.01, paired t-test, Fig. S7
A-B) after ten minutes, and had no effect on blood pressure (7.0 ± 4.6%, not significant, paired
t-test, Fig. S7 B) in Piezo2Cre+;ChR2-eYFP anesthetized mice. We stimulated the superior
laryngeal nerve branch (area 2 as indicated in main Fig. 4A) that generated a significant
light-induced depressor response. Before propranolol administration, optogenetic activation of
Piezo2 sensory nerves induced significant reduction of BP (-46.2 ± 3.2%) and HR (-40.9 ± 4.9%,
Fig S7. C-E). Ten minutes following propranolol injection, the light-induced depressor response
(BP: -19.2 ± 4.0%, HR: -5.3 ± 1.2%, Fig S7. C-E) was significantly attenuated. The small
residual HR response to optogenetic activation is presumably mediated by increased
parasympathetic nerve activity to the heart (Fig. S7 C, D). The light-induced decrease in BP was
attenuated to a lesser extent by propranolol, suggesting that the decrease in HR and cardiac

5
output contributed to the fall in BP (Fig. S7 C, E), while the residual decrease in BP (Fig. S7 C,
E) was primarily mediated by inhibition of peripheral sympathetic activity and vasodilation.
These results clearly suggest sympathoinhibition contributes to the cardiovascular response to
optogenetic activation of Piezo2+ baroreceptor neurons.

6
Fig. S1
SNP-induced unloading of baroreflex and PE-induced phasic/tonic ADN activity in WT
and dKO mice. A, Traces show a SNP-induced baroreflex in WT, but not in dKO mice. B-C,
Statistical analysis of SNP-induced HR and BP changes in WT and dKO mice. D, Traces show
BP and ADN activity induced by PE administration in a WT and a dKO mouse. The phasic ADN
activity was analyzed within 30 seconds following PE injection. Tonic ADN activity was
analyzed over 30 seconds when BP reached a plateau. E, Statistical analysis of phasic and tonic
ADN activities. ***p < 0.001, **p < 0.01, Student’s t-test, means ± s.e.m.

7
Fig. S2
Intact aorta innervation of baroreceptors in Phox2bCre+;Ai9f/+Piezo1f/fPiezo2f/f mice. The
full width of the ventral aortic arch from Phox2bCre+;Ai9f/+ (n=2) or
Phox2bCre+;Ai9f/+Piezo1f/+Piezo2f/+ (n=1) (WT; heterozygous animals show no functional
changes) and Phox2bCre+;Ai9f/+Piezo1f/fPiezo2f/f (dKO) animals was imaged to ascertain
whether knockout animals have appropriate aorta innervation. Baroreceptor innervation spans
one of two regions (green) shown on the aortic arch schematic (left). Individual mice vary
considerably, but WT and dKO mice do not have significantly different innervation density.
Images of aorta innervation from WT and dKO mice are shown (tdTomato, white, 9
z-projections taken at 10×, stitched by Nikon Elements software). Total aorta innervation was
quantified as a fraction of labeled area of a Region of Interest (ROI) that spanned the entire arch.
Fraction of labeled area was then scaled by total ROI size to account for different aorta widths.
Each dot represents one mouse, n=3 mice per group. Scale bar is 200 µm.

8
Fig. S3
Increased BP and urine homovanillic acid level in conscious Phox2bCre+;Piezo1f/fPiezo2f/f
mice. Changes in A, systolic blood pressure B, diastolic blood pressure C, activity and D, urine
homovanillic acid concentration in dKO and WT littermates. #p< 0.05, n.s., statistically not
significant, two-way ANOVA; ***p<0.001, Student’s t-test. Means ± s.e.m.

9
Fig. S4
Representative traces of blood pressure and electrocardiogram radiotelemetry recording.
A, WT. B, dKO mice. Gary areas indicate night period (6pm-6am).

10
Fig. S5
BP variability in conscious nodose/petrosal ganglia-specific Piezo1 conditional knockout
mice. Radiotelemetry recording of A, mean blood pressure B, heart rate and C, activity on
conscious WT and P1cKO mice. Night time is indicated as a gray shading. n.s., statistically not
significant, two-way ANOVA. D, Spontaneous BRS was intact in P1cKO mice (n=8) compared to
WT (n=8). n.s., statistically not significant, Student’s t-test. E, Frequency distribution histogram
of the systolic blood pressure from 72-hour period in WT and P1cKO mice. F, BP variability
reported as standard deviation in WT and P1cKO mice. n.s., statistically not significant, Student’s
t-test. G, The maximum and minimum values of blood pressure in WT and P1cKO mice (n=8). P
values are indicated in the bars. Unpaired Student’s t-test. Means ± s.e.m.

11
Fig. S6
BP variability in conscious nodose/petrosal ganglia-specific Piezo2 conditional knockout
mice. Radiotelemetry recording of A, mean blood pressure B, heart rate and C, activity on
conscious WT and P2cKO mice. Night time is indicated as a gray shading. n.s., statistically not
significant, two-way ANOVA. D, Spontaneous BRS was intact in P2cKO mice (n=7) compared to
WT (n=8). n.s., statistically not significant, Student’s t-test. E, Frequency distribution histogram
of the systolic blood pressure from 72-hour period in WT and P2cKO mice. F, BP variability
reported as standard deviation in WT and P2cKO mice. n.s., statistically not significant, Student’s
t-test. G, The maximum and minimum values of blood pressure in WT and P2cKO mice
(n=10-12). P values are indicated in the bars. Unpaired Student’s t-test. Means ± s.e.m.

12
Fig. S7
Sympathetic inhibition during optogenetic activation of Piezo2 sensory nerves. A-B, Basal
cardiovascular changes before and 10 minutes after intravenous injection of propranolol (5
mg/kg) in the jugular vein in three Piezo2Cre+;ChR2-eYFP mice. **p < 0.01, n.s., statistically
not significant, paired t-test. C, Traces of cardiovascular response following focal vagus nerve
illumination (blue shading) at superior laryngeal nerve branch (area 2) in anesthetized
Piezo2Cre+;ChR2-eYFP mice before and after propranolol injection. D-E, Statistical analysis of
light-induced cardiovascular changes before and after propranolol injection. ***p < 0.001,
Student’s t-test. ### p < 0.001, ## p < 0.01, n.s., statistically not significant, paired t-test. Means
± s.e.m.

13
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