Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Capsaicin induces ablation of TRPV1-lineage axonal terminals. A, schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics). Primary afferent neurons were dissociated from TRPV1-GFP reporter mice and plated in one side of the MFC. Axonal terminals cross the barrier and reach the compartment in the other side (axonal compartment). Capsaicin application was restricted to the axonal compartment, where time-lapse imaging was performed. B—G, representative time-lapse imaging of axonal terminals from MFC. Fluorescence signals from GFP (B–D) and tdTomato (E–G) were monitored before and after the axonal compartment was exposed to capsaicin (100 μm). Scale bar = 10 μm. Arrowheads, ablated fibers; arrow, beaded fibers; asterisk, fibers not affected. Movie files showing the entire field are available (supplemental Movies 1 and 2). H, proportion of unaffected fibers after application of 100 μm capsaicin to either somata (circles) or axonal compartments (comp., squares). Survival proportion is plotted as mean ± S.E. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. Log-rank test; #, p < 0.0001. I, schematic of the multicompartmental culture of DRG neurons dissociated from TRPV1-GFP reporter mice in triple MFC. Time-lapse imaging was performed in axonal compartments A and B before and after application of 100 μm capsaicin. J, proportion of unaffected fibers 28 min after the application of 100 μm capsaicin to compartment A. Survival proportion is plotted as mean ± S.E. Numbers within columns represent the numbers of individual GFP-expressing fibers examined in each group. Log-rank test; #, p < 0.0001.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Imaging, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Capsaicin-induced ablation depends on Ca2+ influx through TRPV1. A and B, in vitro ablation assay in MFC was performed under the indicated conditions. Cap, 100 μm capsaicin; EG, 10 mm EGTA without addition of Ca2+; RR, ruthenium red (a pore blocker of TRPV1), 10 μm; KCl, 50 mm KCl (substituted for 50 mm NaCl); DM, control with DMSO less than 0.1%; Thp, 2 μm thapsigargin (an inhibitor of ER Ca2+ ATPase); Ion, 5 μm ionomycin (a Ca2+ ionophore); Nif, 1 μm nifedipine (an L-type Ca2+ channel blocker). Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. C, quantification and statistical analysis of the experiments in A and B and other conditions. Statistical comparison was performed throughout the entire 28-min time-course by log-rank test, but, for simplicity, only the survival fractions (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment are shown. Cap +, application of 100 μm capsaicin; Cap −, no capsaicin application; Con, control without adding vehicle; A01, 10 μm T16Ainh-A01 (an inhibitor of ANO1, Ca2+-activated Cl− channel); PH, 100 μm 9-phenanthrol (an inhibitor of TRPM4, Ca2+-activated cationic channel). All groups were tested using DRG neurons from TRPV1-lineage reporter mice except TRPV1 KO mice (V1ko). TRPV1 KO mice were tested following electroporation of cDNA encoding GFP into dissociated DRG neurons. Log-rank test; **, p < 0.005; #, p < 0.0001. Numbers within columns represent numbers of axons examined in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: In Vitro, Expressing, Electroporation

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Depolarization of mitochondrial membrane potential, inhibition of mPTP, or ROS do not attenuate capsaicin-induced ablation of TRPV1-lineage nerve terminals. A—C, capsaicin induces Ca2+ loading into mitochondria in a TRPV1-dependent manner in somata of sensory neurons. Shown are representative relative traces of simultaneous recordings of cytosolic (cyto, blue) and mitochondrial (mito, red) Ca2+ levels in dissociated sensory neurons from WT (A) or TRPV1 KO mice (B). A genetically encoded Ca2+-sensing protein targeted to mitochondria (LAR-GECO1.2) was electroporated into dissociated neurons. Ratiometric measurement of Fura-2/AM was performed in the same neurons to assess the cytosolic Ca2+ level. Cap (10 μm) followed by KCl (50 mm) was applied for 10 s at the indicated times. C, averaged Cap-induced changes normalized to the prestimulus baseline. cap(+), WT neurons responding to capsaicin; cap(−), WT neurons not responding to capsaicin. **, p < 0.01; #, p < 0.001 in Student's t test versus WT cap(+). Numbers in parentheses represent the numbers of neurons examined in each group. D, representative traces of whole-cell currents before (black) and after application of 1 μm capsaicin without (blue) or with (red) 10 μm Ru360 in HEK293 cells expressing TRPV1. E, in vitro ablation assay in MFC was performed under the indicated conditions. Cap, 100 μm capsaicin; FOL, 50 nm FCCP + 1 μm oligomycin; FOH, 0.5 μm FCCP + 1 μm oligomycin. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. F, changes in Fura ratio by 10 μm capsaicin following pretreatment of somata of dissociated DRG neurons under the indicated conditions. One-way ANOVA followed by Dunnett's post-hoc test; **, p < 0.005; ***, p < 0.001. Numbers within columns represent the numbers of neurons quantified in each group. G, quantification and statistical analysis of the experiments in E and other conditions. Statistical comparison was performed throughout the entire 28-min time course by log-rank test, but, for simplicity, only the survival fractions (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment are shown. Cap +, application of 100 μm capsaicin; Cap −, no capsaicin application; Con, control without adding vehicle; DM, DMSO; CsA, 3 μm cyclosporin A (an inhibitor of mPTP); TRO, 10 μm TRO19622 (an inhibitor of mPTP); PBN, 2 mm phenyl a-tert-butyl nitrone (a ROS scavenger); AOx, antioxidant supplement (an anti-oxidant mixture with a proprietary composition). Log-rank test; #, p < 0.0001. Numbers within columns represent the numbers of GFP-expressing axons quantified in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Inhibition, Expressing, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Inhibition of calpain attenuates capsaicin-induced ablation. A and B, time course of capsaicin-induced ablation of TRPV1-lineage nerve fibers in the presence of MDL or DMSO (A) or following transfection human calpastatin (Cast) or pcDNA3 (P3, B). Log-rank test; *, p < 0.05; **, p < 0.005. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. C, quantification of the in vitro ablation assay in MFC under the indicated conditions. Survival fraction (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment with three calpain inhibitors are plotted. Veh, DMSO control; CII, 10 μm calpain inhibitor I; MDL, 10 μm MDL28170. Log-rank test; **, p < 0.01. Numbers within columns represent the numbers of axons quantified in each group. D, capsaicin-induced change of Fura ratio in somata of dissociated sensory neurons under the indicated conditions. Numbers within columns represent the numbers of neurons quantified in each group. E, left panel, representative Western blot of calpain 2 (Capn2) in dissociated DRG cultures following treatment with RNAi against calpain 2 or control (Con). Right panel, relative expression of calpain2 in the RNAi-treated group normalized to control. n = 4, p < 0.05, Student's t test. F, time course of capsaicin-induced ablation of TRPV1-lineage nerve fibers following treatment with RNAi against calpain 2. Log-rank test; *, p < 0.05. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Inhibition, Transfection, Expressing, In Vitro, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Capsaicin induces ablation of TRPV1-lineage axonal terminals. A, schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics). Primary afferent neurons were dissociated from TRPV1-GFP reporter mice and plated in one side of the MFC. Axonal terminals cross the barrier and reach the compartment in the other side (axonal compartment). Capsaicin application was restricted to the axonal compartment, where time-lapse imaging was performed. B—G, representative time-lapse imaging of axonal terminals from MFC. Fluorescence signals from GFP (B–D) and tdTomato (E–G) were monitored before and after the axonal compartment was exposed to capsaicin (100 μm). Scale bar = 10 μm. Arrowheads, ablated fibers; arrow, beaded fibers; asterisk, fibers not affected. Movie files showing the entire field are available (supplemental Movies 1 and 2). H, proportion of unaffected fibers after application of 100 μm capsaicin to either somata (circles) or axonal compartments (comp., squares). Survival proportion is plotted as mean ± S.E. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. Log-rank test; #, p < 0.0001. I, schematic of the multicompartmental culture of DRG neurons dissociated from TRPV1-GFP reporter mice in triple MFC. Time-lapse imaging was performed in axonal compartments A and B before and after application of 100 μm capsaicin. J, proportion of unaffected fibers 28 min after the application of 100 μm capsaicin to compartment A. Survival proportion is plotted as mean ± S.E. Numbers within columns represent the numbers of individual GFP-expressing fibers examined in each group. Log-rank test; #, p < 0.0001.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Imaging, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Capsaicin-induced ablation depends on Ca2+ influx through TRPV1. A and B, in vitro ablation assay in MFC was performed under the indicated conditions. Cap, 100 μm capsaicin; EG, 10 mm EGTA without addition of Ca2+; RR, ruthenium red (a pore blocker of TRPV1), 10 μm; KCl, 50 mm KCl (substituted for 50 mm NaCl); DM, control with DMSO less than 0.1%; Thp, 2 μm thapsigargin (an inhibitor of ER Ca2+ ATPase); Ion, 5 μm ionomycin (a Ca2+ ionophore); Nif, 1 μm nifedipine (an L-type Ca2+ channel blocker). Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. C, quantification and statistical analysis of the experiments in A and B and other conditions. Statistical comparison was performed throughout the entire 28-min time-course by log-rank test, but, for simplicity, only the survival fractions (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment are shown. Cap +, application of 100 μm capsaicin; Cap −, no capsaicin application; Con, control without adding vehicle; A01, 10 μm T16Ainh-A01 (an inhibitor of ANO1, Ca2+-activated Cl− channel); PH, 100 μm 9-phenanthrol (an inhibitor of TRPM4, Ca2+-activated cationic channel). All groups were tested using DRG neurons from TRPV1-lineage reporter mice except TRPV1 KO mice (V1ko). TRPV1 KO mice were tested following electroporation of cDNA encoding GFP into dissociated DRG neurons. Log-rank test; **, p < 0.005; #, p < 0.0001. Numbers within columns represent numbers of axons examined in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: In Vitro, Control, Expressing, Comparison, Electroporation

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Depolarization of mitochondrial membrane potential, inhibition of mPTP, or ROS do not attenuate capsaicin-induced ablation of TRPV1-lineage nerve terminals. A—C, capsaicin induces Ca2+ loading into mitochondria in a TRPV1-dependent manner in somata of sensory neurons. Shown are representative relative traces of simultaneous recordings of cytosolic (cyto, blue) and mitochondrial (mito, red) Ca2+ levels in dissociated sensory neurons from WT (A) or TRPV1 KO mice (B). A genetically encoded Ca2+-sensing protein targeted to mitochondria (LAR-GECO1.2) was electroporated into dissociated neurons. Ratiometric measurement of Fura-2/AM was performed in the same neurons to assess the cytosolic Ca2+ level. Cap (10 μm) followed by KCl (50 mm) was applied for 10 s at the indicated times. C, averaged Cap-induced changes normalized to the prestimulus baseline. cap(+), WT neurons responding to capsaicin; cap(−), WT neurons not responding to capsaicin. **, p < 0.01; #, p < 0.001 in Student's t test versus WT cap(+). Numbers in parentheses represent the numbers of neurons examined in each group. D, representative traces of whole-cell currents before (black) and after application of 1 μm capsaicin without (blue) or with (red) 10 μm Ru360 in HEK293 cells expressing TRPV1. E, in vitro ablation assay in MFC was performed under the indicated conditions. Cap, 100 μm capsaicin; FOL, 50 nm FCCP + 1 μm oligomycin; FOH, 0.5 μm FCCP + 1 μm oligomycin. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. F, changes in Fura ratio by 10 μm capsaicin following pretreatment of somata of dissociated DRG neurons under the indicated conditions. One-way ANOVA followed by Dunnett's post-hoc test; **, p < 0.005; ***, p < 0.001. Numbers within columns represent the numbers of neurons quantified in each group. G, quantification and statistical analysis of the experiments in E and other conditions. Statistical comparison was performed throughout the entire 28-min time course by log-rank test, but, for simplicity, only the survival fractions (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment are shown. Cap +, application of 100 μm capsaicin; Cap −, no capsaicin application; Con, control without adding vehicle; DM, DMSO; CsA, 3 μm cyclosporin A (an inhibitor of mPTP); TRO, 10 μm TRO19622 (an inhibitor of mPTP); PBN, 2 mm phenyl a-tert-butyl nitrone (a ROS scavenger); AOx, antioxidant supplement (an anti-oxidant mixture with a proprietary composition). Log-rank test; #, p < 0.0001. Numbers within columns represent the numbers of GFP-expressing axons quantified in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Membrane, Inhibition, Expressing, In Vitro, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: Ca 2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

doi: 10.1074/jbc.M117.778290

Figure Lengend Snippet: Inhibition of calpain attenuates capsaicin-induced ablation. A and B, time course of capsaicin-induced ablation of TRPV1-lineage nerve fibers in the presence of MDL or DMSO (A) or following transfection human calpastatin (Cast) or pcDNA3 (P3, B). Log-rank test; *, p < 0.05; **, p < 0.005. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group. C, quantification of the in vitro ablation assay in MFC under the indicated conditions. Survival fraction (mean ± S.E.) of unaffected GFP-positive fibers 10 min following treatment with three calpain inhibitors are plotted. Veh, DMSO control; CII, 10 μm calpain inhibitor I; MDL, 10 μm MDL28170. Log-rank test; **, p < 0.01. Numbers within columns represent the numbers of axons quantified in each group. D, capsaicin-induced change of Fura ratio in somata of dissociated sensory neurons under the indicated conditions. Numbers within columns represent the numbers of neurons quantified in each group. E, left panel, representative Western blot of calpain 2 (Capn2) in dissociated DRG cultures following treatment with RNAi against calpain 2 or control (Con). Right panel, relative expression of calpain2 in the RNAi-treated group normalized to control. n = 4, p < 0.05, Student's t test. F, time course of capsaicin-induced ablation of TRPV1-lineage nerve fibers following treatment with RNAi against calpain 2. Log-rank test; *, p < 0.05. Numbers in parentheses represent the numbers of individual GFP-expressing fibers examined in each group.

Article Snippet: A , schematic of multicompartmental culture of DRG neurons in an MFC (Xona Microfluidics).

Techniques: Inhibition, Transfection, Expressing, In Vitro, Control, Western Blot

Journal: Scientific Reports

Article Title: Venom composition and pain-causing toxins of the Australian great carpenter bee Xylocopa aruana

doi: 10.1038/s41598-022-26867-8

Figure Lengend Snippet: Pain-causing effects of X. aruana venom components. ( a,b ) Application of XYTX 1 -Xa1a (10 μM) to DRG cells produced an immediate and sustained, non-cell-specific increase in [Ca 2+ ] i . ( c ) Potency of XYTX 1 -Xa1a and melittin in F11 cells, as monitored by changes in [Ca 2+ ] i . ( d–f ) The increase in [Ca 2+ ] i caused by XYTX 1 -Xa1a was potentiated by the presence of venom PLA 2 (1 µM), but not XYTX 2 -Xa2a (1 μM). ** P < 0.01 (unpaired t -test). ( g,h ) Shallow intraplantar injection of XYTX 1 -Xa1a (200 pmol) caused spontaneous pain behaviours in mice which was potentiated by co-injection of venom PLA 2 (20 pmol), but not XYTX 2 -Xa2a (20 pmol). XYTX 2 -Xa2a (20 pmol) alone does not cause spontaneous pain behaviours while venom PLA 2 does (20 pmol). Data are expressed as mean ± SEM ( n = 3–6). ( i ) Co-injection of XYTX 1 -Xa1a (200 pmol) plus venom PLA 2 (20 pmol) caused paw swelling. Data are expressed as mean ± SEM ( n = 3–6). * P < 0.05; ** P < 0.01; **** P < 0.0001 (one way-ANOVA with Tukey’s multiple comparisons).

Article Snippet: F11 (mouse neuroblastoma \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times $$\end{document} × DRG neuron hybrid; European Collection of Authenticated Cell Cultures) were cultured as previously described .

Techniques: Produced, Injection