1. Volume 17, Issue 11, Pages 2901-2912 (December 2016)
TRPV1 Nociceptor Activity Initiates USP5/T-type Channel-Mediated Plasticity 
Patrick Stemkowski, Agustin García-Caballero, Vinicius De Maria Gadotti, Said M'Dahoma, Shuo Huang, Stefanie Alice Gertrud Black, Lina Chen, Ivana Assis Souza, Zizhen Zhang, Gerald Werner Zamponi 
Cell Reports 
Volume 17, Issue 11, Pages (December 2016)
DOI: /j.celrep
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2. Cell Reports 2016 17, 2901-2912DOI: (10.1016/j.celrep.2016.11.047)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1 Cutaneous Nociceptors Are Optogenetically Targeted
(A) Illustration of breeding strategy employed to direct ChR2 to nociceptors in transgenic mice. A TRPV1-cre recombinase driver mouse strain (B6.129-Trpv1tm1(cre)Bbm/J, referred to as TRPV1cre) was crossed with a conditional allele strain (B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4∗H134R/EYFP)Hze/J, referred to as Ai32), where a loxP-flanked STOP cassette prevents downstream expression of ChR2 fused with EYFP (ChR2-EYFP) at the Gt(ROSA)26Sor locus. In offspring, cre recombinase deletes the stop cassette, allowing for ChR2-EYFP expression in TRPV1 lineage neurons (TRPV1-ChR2-EYFP).
(B) Immunohistochemistry (IHC) in DRG cell bodies from TRPV1-ChR2-EYFP mice revealed that ChR2-EYFP (green, left panels) and CGRP (magenta, upper-center panel) co-expressed (white, upper-right panel, white arrows). There was relatively less expression in IB4- (middle panels) and NF200-positive neurons (lower panels).
(C) Quantification in DRG indicated that 43.6% (41/94 cells) ChR2-EYFP neurons co-expressed CGRP, while 25.8% (41/159 cells) and 8.5% (16/188 cells) co-expressed IB4 and NF200, respectively. Quantification of CGRP, IB4 and NF200 labeling was respectively determined from cell body counts in eight, 11, and 13 slices pooled from two animals.
(D) Similar to DRG, IHC in superficial dorsal horn of the spinal cord revealed that ChR2-EYFP (green, left panels) and CGRP (magenta, upper-center panel) co-expressed (white, upper-right panel). There was less expression in IB4-positive neurons (middle panels), and expression in NF200-positive neurons was not detected (lower panels).
(E) IHC in glabrous skin indicated that ChR2-EYFP (green, left panel) and CGRP (magenta, center panel) co-expressed (white, right panel, white arrow).
(F) Representative trace of voltage-clamp recordings from cultured EYFP-positive DRG neurons, showing functional expression of ChR2 in nociceptors, with currents evoked by blue light (wavelength: 470 nm, power: 2 mW, pulse duration: 500 ms) in the same neurons responsive to the TRPV1 channel agonist, capsaicin (10 μM, duration: 7 s).
Scale bars in (B), (D), and (E), 20 μm for DRG, 50 μm for spinal cord, and 20 μm for glabrous skin. Vh, holding potential.
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4. Figure 2
Repetitive Optogenetic Stimulation of Nociceptors Results in Upregulation of USP5 and Cav3.2
(A) WB for USP5 expression in lumbar spinal level 5 (L5) DRG (left panel) revealed that USP5 is upregulated in tissue isolated ipsilateral to stimulation with light (wavelength: 473 nm, irradiance: 10 mW/cm2, rate: 10 Hz, duration: 10 min) in TRPV1-ChR2-EYFP mice. Quantification (right panel) of WB experiments normalized to α-tubulin indicated an approximate 2-fold increase in ipsilateral DRG protein levels (left of break in x axis). A similar upregulation occurred in the ipsilateral spinal dorsal horn at L3–5 (right of break in x axis). All dorsal horn data were normalized to actin controls.
(B) Co-IP of Cav3.2 and USP5 in DRG (left panel) showed that the association of USP5 with Cav3.2 channels is upregulated in ipsilateral tissue. Quantification (right panel) of co-IP experiments normalized to α-tubulin in DRG (left of break in x axis) and actin in spinal dorsal horn (right of break in x axis).
(C) WB for Cav3.2 channel expression in DRG (left panel) revealed that Cav3.2 is upregulated in ipsilateral tissue. Quantification (right panel) of WB experiments in DRG (left of break in x axis) and spinal dorsal horn (right of break in x axis). All data were normalized to actin controls.
Data represent means ± SEM. Values in parentheses indicate sample size (n). Statistical analyses were performed by two-tailed, paired-sample t test; ∗p < 0.05, ∗∗p < WB, western blot; Co-IP, co-immunoprecipitation; IgG, immunoglobulin G; Ipsi, ipsilateral; Contra, contralateral.
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5. Figure 3
Optogenetically Induced Heightening of Pain Responses Rely on USP5-Cav3.2 Interactions
(A) Increased thermal sensitivity was found in the ipsilateral, but not contralateral hind paw at 1 hr post-stimulation.
(B) In a similar manner to (B), only the ipsilateral hind paw became hypersensitive to mechanical stimulation.
(C) Disruption of the interaction between USP5 and Cav3.2 channels with a single injection of Tat-3.2-III-IV peptide (10 μg, i.t.; Group 2) attenuated mechanical hypersensitivity in the ipsilateral hind paw of stimulated mice when compared to sensitized animals that received a control solution, containing Tat-3.2-CT peptide (Group 1). In an analogous manner, mibefradil (Group 3) blocked the development of optically induced mechanical hypersensitivity.
Data represent means ± SEM. Values in parentheses indicate sample size (n). Statistical analyses were performed by repeated-measures two-way ANOVA in (A and B) or one-way ANOVA in (C), followed by a Tukey test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Ipsi, ipsilateral; Contra, contralateral; Stim, stimulation.
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6. Figure 4
Optogenetic Stimulation of Mechanoreceptors Does Not Produce Enhanced USP5 Expression or Hypersensitivity to Mechanical Stimuli
(A) IHC from THY1-ChR2-YFP mice revealed that ChR2-YFP (green, upper panels) and NF200 (magenta, middle-left panels for DRG and spinal cord columns) co-expressed (white) in DRG cell bodies (lower-left panels, white arrows) and dorsal horn of the spinal cord (lower-left panel). There was far less (if any) YFP expression in nociceptive neurons that are positive for CGRP (middle-right panels for DRG and spinal cord columns; middle panel for glabrous skin column) in DRG (lower-right panel), spinal dorsal horn (lower-right panel), as well as in glabrous skin (lower panel; co-incubated with anti-GFP to enhance YFP signal).
(B) Representative trace of voltage-clamp recordings from cultured DRG neurons harvested from THY1-ChR2-YFP mice, showing presence of light-evoked (wavelength: 473 nm; power: 10 mW; pulse duration: 100 ms) ChR2 currents in large diameter, EYFP-expressing cell bodies.
(C) Ipsilateral mechanical withdrawal threshold was not altered at 1 hr post-opto-stimulation (wavelength: 473 nm; irradiance: 10 mW/cm2; rate: 10 Hz; duration: 10 min).
(D) Co-IP blot of Cav3.2 and USP5 in lumbar spinal dorsal horn revealed that the association of USP5 with Cav3.2 channels is not upregulated in tissue isolated ipsilateral to stimulation with light (wavelength: 473 nm, irradiance: 10 mW/cm2, rate: 10 Hz, duration: 10 min) in THY1-ChR2-YFP mice.
(E) Quantification of experiments in (D) normalized to actin.
Data represent means ± SEM. Values in parentheses indicate sample size (n). Statistical analyses were performed by repeated-measures two-way ANOVA, followed by a Tukey test in (C) or two-tailed, paired-sample t test in (E). Scale bars in (A), 20 μm. Vh, holding potential. Ipsi, ipsilateral. Contra, contralateral.
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7. Figure 5
Opto-induced Sensitization to Mechanical Stimuli Involves TRPV1 Nociceptors but Not Mechanoreceptors
(A) Illustration of opto-stimulation in TRPV1-ChR2-EYFP animals, where blue light activates C-fibers, but not A-fibers (left graphic; bright-yellow and dark-black neurons, respectively). Stimulation is immediately followed (arrow) by intraplantar (i.pl.) injections of flagellin (0.3 μg) combined with QX314 (2%) in both hind paws to block A-fiber activity (right graphic; faint black neuron).
(B) Histogram indicates failure of the cocktail to suppress mechanical hypersensitivity, since mechanical withdrawal threshold in the ipsilateral paw was still decreased at 1 hr post-stimulation, relative to before, as well as contralateral values.
(C) An analogous approach to that described in (A), with the exception that a single i.pl. injection of capsaicin (10 μg) mixed with QX314 (2%) was used to silence nociceptive C-fibers in the opto-stimulated (ipsilateral) hind paw (right graphic; faint yellow neuron).
(D) Silencing of nociceptors (Group 2) abolished the effect of opto-stimulation on mechanical threshold, as revealed by a comparison with sensitized mice (Group 1) treated with vehicle (20% ethanol and 5% Tween 20 in saline).
Data represent means ± SEM. Values in parentheses indicate sample size (n). Statistical analyses were performed by repeated-measures two-way ANOVA, followed by a Tukey test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Ipsi, ipsilateral. Contra, contralateral; Stim, stimulation.
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8. Figure 6
Activity-Dependent Sensitization in the Spinal Dorsal Horn Is Mediated by Pre-synaptic T-type Channels
(A) Illustration of in vitro electrophysiological setup in transverse spinal cord slices cut from the lumbar region in TRPV1-ChR2-EYFP mice. This configuration allows for optogenetic stimulation of central projections of nociceptor fibers with an optic cannula (diameter: 400 μm) coupled to a LED (wavelength: 470 nm; power: 2 mW) directed at the dorsal root entry zone, along with recording synaptic activity in lamina II neurons.
(B) Representative trace illustrating EPSCs evoked by light (pulse duration: 50 ms) at a rate of 2 Hz.
(C) Representative traces from the same cell as in (B) depict recordings of spontaneous EPSCs before (top trace) and the overall increase in these events immediately following (bottom trace) opto-stimulation (rate: 2 Hz, duration: 2 min).
(D) Cumulative distribution of spontaneous EPSC IEI from a single representative experiment, indicating the pronounced decrease in IEI and hence facilitation of presynaptic release of excitatory neurotransmitter that follows stimulation of nociceptor fibers with light (KS test, p = 0).
(E) The same experiment as in (D), cumulative distribution of spontaneous EPSC amplitude was altered to a far lesser extent (KS test, p = 0.03).
(F) Quantification of pooled experiments indicated that the percent decrease of IEI in response to opto-stimulation was attenuated when USP5 association with Cav3.2 channels is disrupted in slices pre-treated with Tat-3.2-III-IV peptide (10 μg/mL). No effect was observed in slices pre-treated with control Tat-3.2-CT peptide.
Where applicable, data represent means ± SEM. Values in parentheses indicate sample size (n). Statistical analyses in (F) were performed by one-way ANOVA, followed by a Tukey test; ∗p < Vh, holding potential.
Cell Reports  , DOI: ( /j.celrep )
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