Journal: Frontiers in Physiology

Article Title: 2-Kidney-1-clip hypertension is not attenuated in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) channel

doi: 10.3389/fphys.2025.1713864

Figure Lengend Snippet: Quantification of Mouse Renal Sensory Neurons Expressing Transient Receptor Potential Vanilliod 1 (TRPV1) Channel. (A) Number of retrogradely labelled neurons in the dorsal root ganglion at each thoracic and lumbar spinal segment in both control and 2-kidney-1-clip (2K1C) mice. (B) The percentage of retrogradely labelled neurons that were Trpv1, IB4, or Trpv1 plus IB4 positive. Note, there were no statistical differences in the number of renal sensory neurons or percentage of TRPV1, IB4, and TRPV1+IB4 between sham and 2K1C mice. *P < 0.05 Trpv1 vs. IB4 or TRPV1 plus IB4. (C) Low power image of mouse dorsal root ganglia visualized for Wheat Germ Agglutinin-647 (WGA-647, blue), TRPV1 (red), and IB4 (green). (D-G) Inset images of WGA-647 (blue), TRPV1 (red), IB4 (green), and merged. Unfilled Arrow: TRPV1-positive, IB4- renal sensory neuron; Filled Arrow: TRPV1-negative renal sensory neuron.

Article Snippet: Sections were incubated in a rabbit anti-TRPV1 antibody (1:2,000 at 4 °C for 24 h, Alomone ACC-030) and visualized with donkey anti-rabbit AlexaFluor 555 (1:250 at 4 °C for 24 h, ThermoFisher).

Techniques: Expressing, Control

Journal: Frontiers in Physiology

Article Title: 2-Kidney-1-clip hypertension is not attenuated in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) channel

doi: 10.3389/fphys.2025.1713864

Figure Lengend Snippet: Distribution of Mouse Renal Sensory Neurons as a Function of Cell Diameter Cell diameter was calculated by the average of six measurements per cell and split into 10 um bins. (A) Number of renal sensory neurons in the dorsal root ganglion as a function of cell diameter. (B) Number of TRPV1-positive versus TRPV1-negative renal sensory neurons. (C) Number of IB4+ versus IB4+ plus TRPV1-positive neurons. Values are mean ± SEM and individual data points.

Article Snippet: Sections were incubated in a rabbit anti-TRPV1 antibody (1:2,000 at 4 °C for 24 h, Alomone ACC-030) and visualized with donkey anti-rabbit AlexaFluor 555 (1:250 at 4 °C for 24 h, ThermoFisher).

Techniques:

Journal: Frontiers in Physiology

Article Title: 2-Kidney-1-clip hypertension is not attenuated in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) channel

doi: 10.3389/fphys.2025.1713864

Figure Lengend Snippet: Hemodynamic responses to 2-kidney-1-Clip (2K1C) Hypertension in Wild-Type and Transient Receptor Potential Vanilloid 1 Knockout (Trpv1 −/− ) Male Mice (A) Mean ± SEM of mean, systolic, and diastolic ABP as well as pulse pressure and heart rate in wild-type and Trpv1 −/− mice. Values were averaged between day and night periods. (B) Mean ± SEM and individual data points of the depressor response to ganglionic blockade with chlorisondamine (4 mg/kg, ip) on Day 0 and 23. (C) Mean ± SEM and individual data points of kidney mass for both clipped and unclipped kidneys in wild-type and Trpv1 −/− mice. *P < 0.05 wild-type vs. Trpv1 −/− , #P < 0.05 unclipped vs. clipped. Data were analyzed by a 2-way Mixed ANOVA (strain, time) with repeated measures followed by paired or independent t-tests with layered Bonferroni correction when significant F values were obtained.

Article Snippet: Sections were incubated in a rabbit anti-TRPV1 antibody (1:2,000 at 4 °C for 24 h, Alomone ACC-030) and visualized with donkey anti-rabbit AlexaFluor 555 (1:250 at 4 °C for 24 h, ThermoFisher).

Techniques: Knock-Out

Journal: Frontiers in Physiology

Article Title: 2-Kidney-1-clip hypertension is not attenuated in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) channel

doi: 10.3389/fphys.2025.1713864

Figure Lengend Snippet: Hemodynamic responses to 2-kidney-1-Clip (2K1C) Hypertension in Wild-Type and Transient Receptor Potential Vanilloid 1 Knockout (Trpv1 −/− ) Female Mice. (A) Mean ± SEM of mean, systolic, and diastolic ABP as well as pulse pressure and heart rate in wild-type and Trpv1 −/− mice. Values were averaged between day and night periods. (B) Mean ± SEM and individual data points of the depressor response to ganglionic blockade with chlorisondamine (4 mg/kg, ip) on Day 0 and 23. (C) Mean ± SEM and individual data points of kidney mass for both clipped and unclipped kidneys in wild-type and Trpv1 −/− mice. *P < 0.05 wild-type vs. Trpv1 −/− , #P < 0.05 unclipped vs. clipped. Data were analyzed by a 2-way Mixed ANOVA (strain, time) with repeated measures followed by paired or independent t-tests with layered Bonferroni correction when significant F values were obtained.

Article Snippet: Sections were incubated in a rabbit anti-TRPV1 antibody (1:2,000 at 4 °C for 24 h, Alomone ACC-030) and visualized with donkey anti-rabbit AlexaFluor 555 (1:250 at 4 °C for 24 h, ThermoFisher).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Nociceptors use multiple neurotransmitters to drive pain

doi: 10.1101/2025.09.09.675093

Figure Lengend Snippet: (A-F) . Pain behavioral assessment of Trpv1-Cre DTA and Trpv1-Cre VGLUT2 cKO mice and their respective littermate controls. (A) Latency to withdrawal to radiant heat. n=9 (5M/4F) for Control and n=5 (4M/1F) for DTA; n=9 (4M/5F) for Control and n=11 (6M/5F) for VGLUT2 cKO. (B) Temperature of first lick and jump on Ramping Hot Plate. n=16 (5M/11F) for Control and n=6 (4M/2F) for DTA; n=9 (4M/5F) for Control and n=11 (6M/5F) for VGLUT2 cKO. (C) Latency to withdrawal on Static Hot Plate. n=9 (5M/4F) for Control and n=5 (4M/1F) for DTA; n=18 (11M/7F) for Control and n=9 (6M/3F) for VGLUT2 cKO. (D) Latency to withdrawal to dry ice. n=9 (5M/4F) for Control and n=5 (4M/1F) for DTA; n=9 (4M/5F) for Control and n=11 (6M/5F) for VGLUT2 cKO. (E) Ethogram and summary data showing time spent licking paw injected with capsaicin. n=9 (5M/4F) for Control and n=5 (4M/1F) for DTA; n=5 (3M/2F) for Control and n=7 (5M/2F) for VGLUT2 cKO. (F) Ethogram and summary data showing time spent licking paw injected with mustard oil. n=9 (5M/4F) for Control and n=5 (4M/1F) for DTA; n=10 (3M/7F) for Control and n=12 (5M/7F) for VGLUT2 cKO. Bars represent mean ± SEM. Means were compared by One-WAY ANOVA, with Holm-Sidak multiple comparisons test used to compare DTA and VGLUT2 cKO with one another, and with their respective littermate controls. * p <0.05, ** p <0.01 *** p <0.001.

Article Snippet: Afterward, sections were incubated in blocking solution containing a subset of the following primary antibodies: 1:500 or 1.1000 goat anti-CGRP polyclonal primary antibody (Abcam, #ab36001), 1:500 rat anti-Substance P polyclonal primary antibody (Abcam, #ab67006), 1:500 rabbit anti-TRPV1 polyclonal primary antibody (Alomone Labs, #ACC-030), 1:500 chicken anti-Peripherin polyclonal primary antibody (Invitrogen, #PA1-10012), 1:1000 guinea pig anti-VGlut2 polyclonal primary antibody (Millpore Sigma, #AB2251-I), and 1:1500 rabbit anti-PAM polyclonal primary antibody (Proteintech, #26972) on a shaker at room temperature overnight.

Techniques: Control, Injection

Journal: bioRxiv

Article Title: Nociceptors use multiple neurotransmitters to drive pain

doi: 10.1101/2025.09.09.675093

Figure Lengend Snippet: (A-F) Laser speckle contrast imaging was used to measure the blood flow in the paw following nociceptor stimulation with mustard oil. Images (A) , time course (B) and quantification (area under curve, normalized to control) (C) showing that mustard oil application evokes less blood flow in the paw of PAM cKO mice compared to controls. n=14 (5M/9F) for Control and n=10 (7M/3F) for PAM cKO. This response was completely lost in Trpv1-Cre DTA mice (D-F) . n=12 (5M/7F) for Control and n=6 (4M/2F) for DTA. Bars represent mean ± SEM. Normalized area under the curve was compared using an unpaired t test. * p <0.05, ** p <0.01 *** p <0.001.

Article Snippet: Afterward, sections were incubated in blocking solution containing a subset of the following primary antibodies: 1:500 or 1.1000 goat anti-CGRP polyclonal primary antibody (Abcam, #ab36001), 1:500 rat anti-Substance P polyclonal primary antibody (Abcam, #ab67006), 1:500 rabbit anti-TRPV1 polyclonal primary antibody (Alomone Labs, #ACC-030), 1:500 chicken anti-Peripherin polyclonal primary antibody (Invitrogen, #PA1-10012), 1:1000 guinea pig anti-VGlut2 polyclonal primary antibody (Millpore Sigma, #AB2251-I), and 1:1500 rabbit anti-PAM polyclonal primary antibody (Proteintech, #26972) on a shaker at room temperature overnight.

Techniques: Imaging, Control

Journal: bioRxiv

Article Title: Nociceptors use multiple neurotransmitters to drive pain

doi: 10.1101/2025.09.09.675093

Figure Lengend Snippet: (A) Bulk RNA sequencing shows very few genes are differentially expressed between control and PAM cKO dorsal root ganglia. As expected, Cre was enriched in PAM cKO Trpv1 DRGs, and PAM was downregulated. n=4 (2M/2F) for Control; n=4 (2M/2F) for PAM cKO Trpv1 .

Article Snippet: Afterward, sections were incubated in blocking solution containing a subset of the following primary antibodies: 1:500 or 1.1000 goat anti-CGRP polyclonal primary antibody (Abcam, #ab36001), 1:500 rat anti-Substance P polyclonal primary antibody (Abcam, #ab67006), 1:500 rabbit anti-TRPV1 polyclonal primary antibody (Alomone Labs, #ACC-030), 1:500 chicken anti-Peripherin polyclonal primary antibody (Invitrogen, #PA1-10012), 1:1000 guinea pig anti-VGlut2 polyclonal primary antibody (Millpore Sigma, #AB2251-I), and 1:1500 rabbit anti-PAM polyclonal primary antibody (Proteintech, #26972) on a shaker at room temperature overnight.

Techniques: RNA Sequencing, Control

Journal: bioRxiv

Article Title: Nociceptors use multiple neurotransmitters to drive pain

doi: 10.1101/2025.09.09.675093

Figure Lengend Snippet: (A) Example confocal images of spinal cord dorsal horns showing that PAM deletion reduces the intensity of CGRP and Substance P (SP) staining in the terminal field of Trpv1 afferents. The dotted lines are traced from the region of Trpv1 staining. Note that only in the area of Trpv1 innervation would we expect to see reduced neuropeptide staining using the Trpv1-Cre approach.

Article Snippet: Afterward, sections were incubated in blocking solution containing a subset of the following primary antibodies: 1:500 or 1.1000 goat anti-CGRP polyclonal primary antibody (Abcam, #ab36001), 1:500 rat anti-Substance P polyclonal primary antibody (Abcam, #ab67006), 1:500 rabbit anti-TRPV1 polyclonal primary antibody (Alomone Labs, #ACC-030), 1:500 chicken anti-Peripherin polyclonal primary antibody (Invitrogen, #PA1-10012), 1:1000 guinea pig anti-VGlut2 polyclonal primary antibody (Millpore Sigma, #AB2251-I), and 1:1500 rabbit anti-PAM polyclonal primary antibody (Proteintech, #26972) on a shaker at room temperature overnight.

Techniques: Staining

Journal: bioRxiv

Article Title: Nociceptors use multiple neurotransmitters to drive pain

doi: 10.1101/2025.09.09.675093

Figure Lengend Snippet: Example confocal images showing spinal cord dorsal horn staining for Trpv1 and cholera toxin B injected into the paw in Control (top) and PAM VGLUT2 cDKO mice (bottom).

Article Snippet: Afterward, sections were incubated in blocking solution containing a subset of the following primary antibodies: 1:500 or 1.1000 goat anti-CGRP polyclonal primary antibody (Abcam, #ab36001), 1:500 rat anti-Substance P polyclonal primary antibody (Abcam, #ab67006), 1:500 rabbit anti-TRPV1 polyclonal primary antibody (Alomone Labs, #ACC-030), 1:500 chicken anti-Peripherin polyclonal primary antibody (Invitrogen, #PA1-10012), 1:1000 guinea pig anti-VGlut2 polyclonal primary antibody (Millpore Sigma, #AB2251-I), and 1:1500 rabbit anti-PAM polyclonal primary antibody (Proteintech, #26972) on a shaker at room temperature overnight.

Techniques: Staining, Injection, Control