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

Figures

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  Figure 1. TrkC expression in peripheral ganglia (A–F) Immunofluorescent staining of DRG sections from TrkCCreERT2::Rosa26ChR2-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).…

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  Figure 2. TrkC mediated recombination in peripheral tissue (A–D) A whole-mount skin preparation from a TrkCCreERT2::Rosa26ChR2-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 TrkCCreERT2::Rosa26ChR2-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 TrkCCreERT2::Rosa26ChR2-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.

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  Figure 3. Loss of function in TrkC+ neurons (A) Survival rate of TrkCCreERT2AviliDTR (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 TrkCCreERT2::AviliDTR::Rosa26ChR2-YFP mice after DTX injection. Scale bars, 50 μm. (D and E) Representative LSCI images of the ear of a control (D) and a TrkCCreERT2::AviliDTR 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.

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  Figure 4. Ablation of TrkC+ neurons leads to blood pressure and heart rate alterations (A) Upon systemic injection of DTX, TrkCCreERT2::AviliDTR 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 TrkCCreERT2::AviliDTR 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 TrkCCreERT2::AviliDTR 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 TrkCCreERT2::AviliDTR and control mice.

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  Figure 5. Activation of TrkC+ neurons (A) Schematic showing in vivo circuit activated by CNO in TrkCCreERT2::AvilhM3Dq mice, or 488 nm of light in TrkCCreERT2::Rosa26ChR2-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 TrkCCreERT2::AvilhM3Dq 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 TrkCCreERT2::Rosa26ChR2-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…

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  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 TrkCCreERT2::AvilhM3Dq 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) TrkCCreERT2::AvilhM3Dq 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 TrkCCreERT2::AvilhM3Dq 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 TrkCCreERT2::AvilhM3Dq 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…

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  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) TrkCCreERT2::AvilhM3Dq 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 TrkCCreERT2::AvilhM3Dq 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 TrkCCreERT2::AvilhM3Dq (red symbols) and control mice (black symbols) treated with CNO.