Identification of a population of peripheral sensory neurons that regulates blood pressure

doi:10.1016/j.celrep.2021.109191

. 2021 Jun 1;35(9):109191.

doi: 10.1016/j.celrep.2021.109191.

Identification of a population of peripheral sensory neurons that regulates blood pressure

Chiara Morelli¹, Laura Castaldi², Sam J Brown³, Lina L Streich⁴, Alexander Websdale³, Francisco J Taberner⁵, Blanka Cerreti³, Alessandro Barenghi³, Kevin M Blum⁶, Julie Sawitzke³, Tessa Frank³, Laura K Steffens⁷, Balint Doleschall³, Joana Serrao³, Denise Ferrarini³, Stefan G Lechner⁸, Robert Prevedel⁹, Paul A Heppenstall¹⁰

Affiliations

¹ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; Collaboration for joint PhD degree between EMBL Heidelberg, Heidelberg, Germany, and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany.
² EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany.
³ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy.
⁴ Collaboration for joint PhD degree between EMBL Heidelberg, Heidelberg, Germany, and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
⁵ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.
⁶ Center for Regenerative Medicine, the Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA; Department of Biomedical Engineering, the Ohio State University, Columbus, OH, USA.
⁷ Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
⁸ Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.
⁹ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
¹⁰ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany. Electronic address: [email protected].

PMID: 34077727
PMCID: PMC8187988
DOI: 10.1016/j.celrep.2021.109191

Identification of a population of peripheral sensory neurons that regulates blood pressure

Chiara Morelli et al. Cell Rep. 2021.

. 2021 Jun 1;35(9):109191.

doi: 10.1016/j.celrep.2021.109191.

Authors

Affiliations

¹ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany; Collaboration for joint PhD degree between EMBL Heidelberg, Heidelberg, Germany, and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany.
² EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany.
³ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy.
⁴ Collaboration for joint PhD degree between EMBL Heidelberg, Heidelberg, Germany, and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
⁵ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.
⁶ Center for Regenerative Medicine, the Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA; Department of Biomedical Engineering, the Ohio State University, Columbus, OH, USA.
⁷ Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
⁸ Institute of Pharmacology, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.
⁹ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; Developmental Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
¹⁰ EMBL Rome, Via Ramarini 32, Monterotondo 00015, Italy; Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany. Electronic address: [email protected].

PMID: 34077727
PMCID: PMC8187988
DOI: 10.1016/j.celrep.2021.109191

Abstract

The vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons, marked by tropomyosin receptor kinase C (TrkC) and tyrosine hydroxylase in the dorsal root ganglia, which projects to blood vessels. Local stimulation of TrkC neurons decreases vessel diameter and blood flow, whereas systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Ablation of the neurons provokes variability in local blood flow, leading to a reduction in systolic blood pressure, increased heart rate variability, and ultimately lethality within 48 h. Thus, a population of TrkC⁺ sensory neurons forms part of a sensory-feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system.

Keywords: DRG; TH; TrkC; blood pressure; cardiovascular homeostasis; peripheral nervous system.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1**
TrkC expression in peripheral ganglia (A–F) Immunofluorescent staining of DRG sections from TrkC^CreERT2::Rosa26^ChR2-YFP (yellow) showed that 9.6% of DRG neurons co-express markers of proprioceptors (Pvalb, red; NF200, magenta) (A and F) and 11.6% of slowly adapting (SA) field mechanoreceptors (Ret, green; NF200, red) (B and F). Very few TrkC⁺ neurons overlap with markers of non-peptidergic nociceptors (IB4, red) (C and F) or peptidergic nociceptors (CGRP, blue) (D and F); 7% of all DRG neurons are also positive for Th (red), a marker of C-fiber low-threshold mechanoreceptors (C-LTMRs) (E and F). (F) Quantification of TrkC co-expression with the other markers in DRGs showing the percentage of total neurons. (G) TrkC⁺/Th⁺ DRG neurons are significantly larger than TrkC⁻/Th⁺ neurons and smaller than TrkC⁺/Th⁻ neurons (298 neurons from three mice). ^∗p < 0.001. (H) Quantification of the number of DRG neurons co-expressing TrkC and Th at different spinal segmental levels, expressed as the percentage of the total number of DRG neurons (n = 3). (I and J) TrkC is not expressed in the nodose-petrosal-jugular ganglion complex (I) and is not expressed in the superior cervical ganglion (J). (K) Left, sketch depicting the mechanical-stimulation protocol and an example trace of a mechanically activated current (MA current) in a small TrkC⁺ neuron. Right, distribution of the different types of MA currents in the small TrkC⁺ neurons (24 neurons from three mice): rapidly adapting (RA), intermediate adapting (IA), and non-responding (NR). (L) Left, example responses to capsaicin and pH 5.4 in a small TrkC⁺ neuron. Right, number of neurons responding (R) and non-responding (NR) to capsaicin or pH in small TrkC⁺ neurons. Arrows indicate co-expression of TrkC⁺ neurons with the other markers. Scale bars, 50 μm.

**Figure 2**
TrkC mediated recombination in peripheral tissue (A–D) A whole-mount skin preparation from a TrkC^CreERT2::Rosa26^ChR2-YFP mouse shows TrkC⁺ Aβ field mechanoreceptors (arrowheads) and TrkC⁺ cells surrounding blood vessels (arrows). (B–D) TrkC is expressed in perivascular cells throughout the body: skin (B), aorta (C), and saphenous artery (D). Mice were injected with Evans blue (EB) i.v. to visualize blood vessels; the saphenous nerve is also evident in (D). (E) Immunohistochemical analysis with anti-desmin antibodies in TrkC^CreERT2::Rosa^26ChR2-YFP mice reveals that TrkC marks a population of desmin-positive perivascular cells. (F) Immunostaining with anti-α-SMA antibodies indicates that TrkC⁺ cells are a subpopulation of vSMC. (G) The vasculature is innervated by TrkC⁺ neurons (arrowheads). TrkC⁺ perivascular cells are also indicated (arrows). (H and I) Intrathecal injection of 45 ng of 4-hydroxytamoxifen in TrkC^CreERT2::Rosa26^ChR2-YFP mice leads to robust expression of YFP in DRGs (H) and in neurons innervating blood vessels (I) but not in TrkC⁺ vSMC (I). TrkC⁺ neurons innervating the vessels co-express the marker Th (I). Scale bars, 50 μm.

**Figure 3**
Loss of function in TrkC⁺ neurons (A) Survival rate of TrkC^CreERT2Avil^iDTR (red symbols, n = 10) and control mice (black symbols, n = 10) upon intraperitoneal administration of DTX. (B and C) Ablation leads to a complete absence of TrkC⁺ neurons in DRG and of TrkC⁺/Th⁺ fibers innervating blood vessels. Immunofluorescence with Th antibodies on DRG sections (B) and whole-mount skin (C) from TrkC^CreERT2::Avil^iDTR::Rosa26^ChR2-YFP mice after DTX injection. Scale bars, 50 μm. (D and E) Representative LSCI images of the ear of a control (D) and a TrkC^CreERT2::Avil^iDTR mouse (E) before DTX injection (0) and at 16, 24, and 32 h after treatment. Scale bars, 1 mm. (F) Measurements of the vascularized area showed blood flow alteration upon ablation of TrkC⁺ neurons. ^∗p < 0.05.

**Figure 4**
Ablation of TrkC⁺ neurons leads to blood pressure and heart rate alterations (A) Upon systemic injection of DTX, TrkC^CreERT2::Avil^iDTR mice display a decrease in blood pressure (red symbols, n = 5), whereas control mice (black symbols, n = 6) are not affected. ^∗p < 0.001. (B) Average heart rate in TrkC^CreERT2::Avil^iDTR mice (red symbols, n = 5) after DTX injections at different time points over 30 min compared with control mice (black symbols, n = 6). (C and D) Fluctuations in heart rate for individual mice over 30 min at 24 (C) and 32 (D) h after DTX injection. (E) Long-term heart rate variability derived from the length of the major (SD2) axis of Poincaré plots at different time points after injection of DTX. Significant differences in variability are evident in TrkC^CreERT2::Avil^iDTR mice compared with controls at 24 and 32 h after injection. ^∗p < 0.05. (F) Short-term variability derived from the length of the minor (SD1) axis of the Poincaré plots at different time points after injection of DTX in TrkC^CreERT2::Avil^iDTR and control mice.

**Figure 5**
Activation of TrkC⁺ neurons (A) Schematic showing *in vivo* circuit activated by CNO in TrkC^CreERT2::Avil^hM3Dq mice, or 488 nm of light in TrkC^CreERT2::Rosa26^ChR2-YFP mice injected intrathecally with 45 ng of 4-hydroxytamoxifen. (B) Representative LSCI images showing blood vessels in the hind paw of control mice (top panels) or TrkC^CreERT2::Avil^hM3Dq mice (bottom panels) treated locally with CNO imaged before and 30 min after the treatment. Scale bars, 1 mm. (C) Measurements of the vascularized area of LSCI images showing blood flow reduction upon local activation of TrkC⁺ neurons with CNO (red symbols, ^∗p < 0.001, n = 3). (D) Three-photon volumetric image of blood vessels in TrkC^CreERT2::Rosa26^ChR2-YFP mouse ear labeled with dextran-fluorescein. Maximum-intensity projection along the lateral imaging plane (x/y) and orthogonal projection of three-dimensional (3D) volume along lines indicated in the (x/y) image by the white, dotted line. Image of green-labeled artery was acquired before the optogenetic stimulus, and red was after the stimulus. Scale bar, 25 μm. (E) Quantification of blood vessel diameter after optical stimulation. ^∗p < 0.005. (F) Three-photon image of blood vessels in the mouse ear labeled with dextran-fluorescein. Red line indicates the laser scan path used to measure and calculate red blood cell velocity. Line-scans were acquired at 666 Hz. (G) Top, line scans generated from the path indicated by the red line in (F) can be stacked as a space-time (x/t) plot, in which the apparent angle is proportional to flow velocity. The images show ~500 ms of data collection before and after optogenetic stimulation, respectively. Bottom, red blood cell velocity before and after stimulation. Velocity was calculated based on radon analysis of spatially windowed space-time plot data (window size, 50 ms). (H) Schematic showing *ex vivo* stimulation of TrkC⁺ afferents (blue) by CNO in TrkC^CreERT2::Avil^hM3Dq mice or 488 nm of illumination in TrkC^CreERT2::Rosa26^ChR2-YFP mice injected intrathecally or *ex vivo* stimulation of vSMC (orange) by norepinephrine (NE) or 488 nm light in TrkC^CreERT2::Rosa26^ChR2-YFP mice injected systemically. (I) Representative images of blood vessels from an *ex vivo* skin-nerve preparation before and 14 min after optogenetic activation of TrkC⁺ neurons (top panels) or application of CNO (bottom panels). Scale bars, 25 μm. (J) Quantification of blood vessel diameter after *ex vivo* stimulation of TrkC⁺ afferents with 488 nm of light or CNO application. (K) Representative images of blood vessels before and 14 min after optogenetic activation of vSMC (top panels) or application of norepinephrine (bottom panels). Dotted lines indicate blood vessel perimeter; arrows indicate shrinkage. Scale bars, 25 μm. (L) Quantification of blood vessel diameter after *ex vivo* stimulation of TrkC⁺ vSMC with 488 nm or light or norepinephrine application. ^∗p < 0.01.

**Figure 6**
Systemic chemogenetic activation of TrkC⁺ neurons leads to blood pressure and heart rate alterations (A) Schematic showing circuit activated by C21 *in vivo*. (B) Systemic injection of C21 in TrkC^CreERT2::Avil^hM3Dq mice leads to elevated blood pressure (red symbols, n = 6) compared with control mice (black symbols, n = 6). ^∗p < 0.01. This is reverted by co-administration of propranolol (yellow symbols, n = 6). (C) TrkC^CreERT2::Avil^hM3Dq mice (red symbols, n = 6) treated systemically with C21 display increased average heart rate compared with mice co-treated with propranolol (yellow symbols, n = 6) or control mice (black and green symbols). (D) Fluctuations in heart rate in individual mice are apparent in TrkC^CreERT2::Avil^hM3Dq mice treated with C21 (red lines) but not in mice co-treated with propranolol (yellow lines) or control animals (black and green lines). (E) Long-term heart rate variability derived from the length of the major (SD2) axis of Poincaré plots in TrkC^CreERT2::Avil^hM3Dq mice treated with C21, C21 plus propranolol, or in control mice. ^∗p < 0.05. (F) Short-term heart rate variability derived from the length of the minor (SD1) axis of Poincaré plots in TrkC^CreERT2::Avil^hM3Dq mice treated with C21, C21 plus propranolol, or in control mice.

**Figure 7**
Behavioral responses to mechanical and thermal stimulation upon chemogenetic activation of TrkC⁺ sensory neurons (A) Schematic of behavioral tests performed after local injection of CNO. (B and C) TrkC^CreERT2::Avil^hM3Dq developed hypersensitivity to punctate mechanical stimuli (n = 6) ^∗p < 0.05 (B), which started 10 min after CNO local treatment and persisted for more than 40 min (C) (0.02 g filament, n = 5). ^∗p < 0.05. (D) Local intraplantar injection of CNO in TrkC^CreERT2::Avil^hM3Dq mice does not provoke mechanical hypersensitivity to dynamic mechanical stimuli (paintbrush, n = 6). (E and F) Thermal sensitivity as assayed by the acetone drop test (n = 6) (E) and hot-plate test (n = 5) (F) was not different between TrkC^CreERT2::Avil^hM3Dq (red symbols) and control mice (black symbols) treated with CNO.

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References

1. Abrahamsen B., Zhao J., Asante C.O., Cendan C.M., Marsh S., Martinez-Barbera J.P., Nassar M.A., Dickenson A.H., Wood J.N. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science. 2008;321:702–705. - PubMed
1. Adelson D.W., Wei J.Y., Kruger L. Warm-sensitive afferent splanchnic C-fiber units in vitro. J. Neurophysiol. 1997;77:2989–3002. - PubMed
1. Albrecht P.J., Hou Q., Argoff C.E., Storey J.R., Wymer J.P., Rice F.L. Excessive peptidergic sensory innervation of cutaneous arteriole-venule shunts (AVS) in the palmar glabrous skin of fibromyalgia patients: implications for widespread deep tissue pain and fatigue. Pain Med. 2013;14:895–915. - PubMed
1. Anitescu M. Ischemic pain. In: Cheng J., Rosenquist R.W., editors. Fundamentals of Pain Medicine. Springer International Publishing; 2018. pp. 141–151.
1. Arcourt A., Gorham L., Dhandapani R., Prato V., Taberner F.J., Wende H., Gangadharan V., Birchmeier C., Heppenstall P.A., Lechner S.G. Touch receptor-derived sensory information alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron. 2017;93:179–193. - PubMed

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[1] Abrahamsen B., Zhao J., Asante C.O., Cendan C.M., Marsh S., Martinez-Barbera J.P., Nassar M.A., Dickenson A.H., Wood J.N. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science. 2008;321:702–705. - PubMed

[2] Abrahamsen B., Zhao J., Asante C.O., Cendan C.M., Marsh S., Martinez-Barbera J.P., Nassar M.A., Dickenson A.H., Wood J.N. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science. 2008;321:702–705. - PubMed

[3] Adelson D.W., Wei J.Y., Kruger L. Warm-sensitive afferent splanchnic C-fiber units in vitro. J. Neurophysiol. 1997;77:2989–3002. - PubMed

[4] Adelson D.W., Wei J.Y., Kruger L. Warm-sensitive afferent splanchnic C-fiber units in vitro. J. Neurophysiol. 1997;77:2989–3002. - PubMed

[5] Albrecht P.J., Hou Q., Argoff C.E., Storey J.R., Wymer J.P., Rice F.L. Excessive peptidergic sensory innervation of cutaneous arteriole-venule shunts (AVS) in the palmar glabrous skin of fibromyalgia patients: implications for widespread deep tissue pain and fatigue. Pain Med. 2013;14:895–915. - PubMed

[6] Albrecht P.J., Hou Q., Argoff C.E., Storey J.R., Wymer J.P., Rice F.L. Excessive peptidergic sensory innervation of cutaneous arteriole-venule shunts (AVS) in the palmar glabrous skin of fibromyalgia patients: implications for widespread deep tissue pain and fatigue. Pain Med. 2013;14:895–915. - PubMed

[7] Anitescu M. Ischemic pain. In: Cheng J., Rosenquist R.W., editors. Fundamentals of Pain Medicine. Springer International Publishing; 2018. pp. 141–151.

[8] Anitescu M. Ischemic pain. In: Cheng J., Rosenquist R.W., editors. Fundamentals of Pain Medicine. Springer International Publishing; 2018. pp. 141–151.

[9] Arcourt A., Gorham L., Dhandapani R., Prato V., Taberner F.J., Wende H., Gangadharan V., Birchmeier C., Heppenstall P.A., Lechner S.G. Touch receptor-derived sensory information alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron. 2017;93:179–193. - PubMed

[10] Arcourt A., Gorham L., Dhandapani R., Prato V., Taberner F.J., Wende H., Gangadharan V., Birchmeier C., Heppenstall P.A., Lechner S.G. Touch receptor-derived sensory information alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron. 2017;93:179–193. - PubMed

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Identification of a population of peripheral sensory neurons that regulates blood pressure

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Identification of a population of peripheral sensory neurons that regulates blood pressure

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