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Supplementary Figures 1 2 Supplementary Figure 1: TRPV1 expression in the ACC of both naïve and CCI 3 young mice. 4

5 As in adult mice, the anti-TRPV1 MAb (green label) stains cortical microglial 6 processes and somas (red label) of naïve young mice, as shown by the colocalization 7 (in yellow) between the anti-TRPV1 and the iba-1. 8 Unlike CCI adult animals, TRPV1 is purely expressed in the microglia of the ACC of 9 1-week CCI young mice (4 experiments from 4 mice, n=4/4). Considering the faster 10 cellular reproduction in youngs, TRPV1 expression has been tested in the ACC 11 sections from 2-weeks CCI young mice. Also at this time point, TRPV1 is selectively 12 expressed in microglia cells (n=2/2). Same result is observed in the ACC of 3 days 13 CCI young mice (data not shown). 14 15

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Supplementary Figure 2: TRPV1 antibody validation. 16

17 18 High resolution confocal laser scanning photomicrographs of ACC sections from the 19 same wt (a-c) and the same TRPV1-/- mice (d-f), showing the staining pattern of 20 three different anti-TRPV1 antibodies (Abs). (a) Anti-TRPV1 MAb labeling of fibers 21 (Millipore Chemicon, 1:100). Same protocol of immunostaining on TRPV1-/- mice 22 tissues (d) shows no stain from both primary or secondary antibody (the latter tested 23 on the background controls of secondary Ab2s; data not shown). (b,c) The cell 24 cytoplasm or whole-cell body labelling of two anti-TRPV1 pAbs is similar in cortical 25 tissues from wt and -/- mice (e, Immunological Sciences, 1:100; f. Neuromics, 1:500). 26 Scale bar, 15 μm. 27 28 Supplementary Figure 3: Both Iba-1 and TRPV1 signals were higher in young 29 than adult mice. 30 31

32 Quantification of the area filled by the TRPV1 signal and Iba-1 one. In cortical 33 sections from naïve young mice both TRPV1 (n=12/8) and Iba-1 (7/6) signal areas are 34 TR

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significantly larger compare adult animals (n=16/9 for TRPV1, n=6/6 for Iba-1; 35 *p<0.05). 36 37 Supplementary Figure 4: Expression of TRPV1 mRNA and protein in microglial 38 cells, neurons and mouse tissue 39

40 (a) RT-PCR of TRPV1 gene expression in cortical neurons, microglial cells and 41 spinal cord isolated from C57BL6J or TRPV1-/- mice: for each sample, TRPV1 42 threshold cycle was normalized to that of β-actin. Data are expressed as mean±SD and 43 are representative of four independent pools of mouse cortices (3-4 mice/pool) 44 (**p<0.01 one-way ANOVA for left panel and *p<0.05 and **p<0.001 Student t-test 45

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for the remnant panels). A representative agarose gel (2%) electrophoresis of two 46 samples per cell type/tissue is also shown in order to display the specificity of RT-47 PCR reactions. (b) Representative immunoblot of TRPV1 expression in ACC from 48 wild-type (wt) and TRPV1-/- mice. Protein lysate of each tissue was subjected to 49 immunoblotting following SDS-10% PAGE against the anti-TRPV1 monoclonal 50 antibody. The bottom portion of the nitrocellulose membranes was probed with the 51 anti–actin antibody, as loading control. N.S.: non specific band. Note that this band is 52 still present in tissue from TRPV1-/- mice. (c) Representative immunoblots comparing 53 the TRPV1 content in ACC (50 µg) and microglial cells (6 µg). Microglial cells 54 isolated from mice were lysed and subjected to immunoblotting against the anti-55 TRPV1 antibody. Note that N.S. band is absent in microglia cell cultures. Due to a 56 strong difference in relative abundance of actin in the protein extracts of ACC tissues 57 and isolated microglial cells (data not shown), the enrichment in TRPV1 expression 58 was assessed by normalizing optical density values for the Ponceau staining of total 59 proteins74. Particularly, optical density values were internally normalized by Ponceau 60 staining of total protein content and further corrected for the value of ACC considered 61 equal to 1. *p<0.05 vs ACC. The purity of microglial cultures was assessed by 62 assessing specific markers for neurons (NeuN) and microglia cells (Iba1). (d) Bar 63 graph of summary data of normalized densitometric ratios ± S.D. of three separate 64 experiments. (e) Left, Current-voltage relationship of the capsaicin-induced current by 65 application of capsaicin 1μM in a microglial cell from acute cortical slice of 66 CX3CR1+/GFP mouse before (black curve), during (red curve) and after (blue curve) 67 capsaicin application; Middle, capsaicin induces an outward rectifying current with 68 reversal potential at about 0 mV (results obtained by subtracting the current before 69 and after the capsaicin application). Right, time plot of the mean current amplitude at 70 +50 mV (n=9). The arrow indicate 3-9 minutes of capsaicin application. 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

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Supplementary Figure 5: TRPV1 distribution in astrocytes of the ACC and 86 hippocampus of adult naïve mice. 87

88 89 (a,d) Representative photographs of anti-TRPV1 and anti-GLAST (a,b) or anti-GLT1 90 (c,d) positive astrocytes of ACC (a,c) and hippocampus (HIPPO; b,d). Merging areas 91 magnifications are presented in the middle panels. Scale bar, 60μm. In the right 92 panels, correlative color scatter plots of TRPV1 (green) and GLAST/ GLT1 (red) for 93 the single experiment reported in a,b,c,and d, show the colocalization (yellow) of the 94 two antibodies (in a, r= 0.046; in b, r= 0.17; in c, r= 0.043; in d r= 0.194). 95 96 97 98 99 100 101

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Supplementary Figure 6: TRPV1 is distributed in the microglia of other brain 102 areas. 103 104

105 106 (a-d) Confocal laser scanning photomicrographs of somatosensory cortex (SS n=17/9), 107 dentate gyrus (DG n=17/9), thalamus (THAL n=17/9) and periaqueductal gray (PAG 108 n=11/6) showing labeling for anti-TRPV1, Iba-1 and their merged. Like the ACC, 109 also in these brain regions the TRPV1 is mainly expressed in the microglia. Scale bar, 110 60μm for the lower magnification images. 111 112 113 114 115 116 117

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Supplementary Figure 7: In the dorsal horn of naïve mice TRPV1 is also 118 expressed in neurons. 119

120 121 Representative confocal images of spinal cord dorsal horn sections immunolabeled 122 with the TRPV1 Mab (green), anti- GFAP, CD11b, and NeuN (all red) from WT 123 (a,b,c) and TRPV1-/- mice (d). Differently from brain areas, TRPV1 is also present in 124 neurons of spinal cord naïve mice (n=4/4). Scale bar, 30μm for the lower 125 magnification images. 126 127 128 129 130 131 132 133 134

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Supplementary Figure 8: TRPV1 is expressed in both glutamatergic and 135 GABAergic neurons of the dorsal and ventral horn 136

137 (a,b)Photomicrographs of immunofluorescence for the anti-TRPV1 MAb (green) and 138 anti-CAMKII (red) in dorsal (DH) and ventral horn (VH). CAMKII is highly and 139 diffusely expressed in DH and less in VH of naïve animal spinal cord. The anti-140 TRPV1 MAb is highly expressed and partially co-localizes with CAMKII present in 141 body cells of both horns (merged panels-yellow). (b,c) Photomicrographs of double 142 immunofluorescence for anti-GAD65/67 (red) and anti-TRPV1 MAb (green). The 143 GAD65/67 is stained as dot-like pattern in DH and VH of naïve animal spinal cord. 144 The anti-TRPV1 MAb is highly expressed and partially co-localizes with the anti-145 GAD65/67 present in body cells (merged panels - yellow). 146 Note that the TRPV1 expression on VH neurons may provide a preliminary evidence 147 on a possible involvement of TRPV1 signaling in the nocifensive behavior. 148 149 150 151 152

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Supplementary Figure 9: anti-TRPV1 MAb staining pattern in adult WT and 153 TRPV1-/- DRG neurons 154

155 40x confocal images (left panels) and detail magnification (right panels) of DRG 156 sections. Double staining of anti-TRPV1 MAb (green) with anti-CGRP (a), anti-IB4 157 (b) anti-NF200 (c, all in red) in DRG neurons of WT animals (n=4) indicates that part 158 of neurons identified into each subgroup expressed TRPV1 (yellow; 32% of CGRP+ 159 neurons are TRPV1+, 14.5 % of IB4+ neurons are TRPV1+, 14 % NF100+ cells were 160 TRPV1+). (d) In TRPV1-/- DRG no cells show labelling for TRPV1 MAb (n=3). Scale 161 bar, 60μm for the lower magnification images. 162 Note that the NF200+ neurons might correspond to the heat-sensitive myelinated 163 nociceptors90. 164

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Supplementary Figure 10: TRPV1 distribution pattern in 3 days CCI adult mice. 165 166

167 Double immunostaining for (a) TRPV1 (in green) and neurons (purple label), and for 168 (b) TRPV1 and microglial cells (in red) from ACC sections of 3 days CCI mice. Anti-169 TRPV1 positive fibers surround neuronal bodies and are fully labeled with the iba-170 1(merging points in yellow). The TRPV1 expression pattern in the cortex of 3 days 171 CCI mice were similar to naïve mice (see Fig 1). Scale bar, 60μm for the lower 172 magnification images. 173

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Supplementary Figure 11: Anti-TRPV1 neuronal labeling was absent in CCI 174 TRPV1-/- mice. 175

176 Confocal laser scanning photomicrographs of ACC sections containing superficial 177 layers from naïve (a), 1 week (b) and 1 month CCI TRPV1-/- mice (c,d) stained with 178 the anti-TRPV1 MAb (Millipore Chemicon, 1:100) (a-c) or background control of 179 secondary Ab (d). Anti-TRPV1 MAb shows no staining in naïve condition (a, n=4) 180 and neither at 1 week (b, n=3) nor at 4 weeks (c, n=6) after CCI. Background control 181 of secondary Ab shows no false staining (d). 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196

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Supplementary Figure 12: Capsaicin-induced increase of mEPSC is due to 197 AMPA receptors activation 198

199 (a) The non-NMDA receptors antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 200 20 μM) completely abolished both frequency and amplitude of mEPSCs (n= 6, paired 201 T-test p= <0.001;). In these conditions, capsaicin increased neither frequency nor 202 amplitude of mEPSCs, suggesting that downstream TRPV1 signalling is AMPA 203 receptor mediated. The NMDA component was excluded in this type of experiments 204 (resting membrane potential -70mV, [Mg2+] 2mM in the internal solution). (b,c) Glia-205 derived glutamate facilitates neurotransmitters release through presynaptic 206 metabotropic receptors (Fiacco and McCarthy, 2004;Perea and Araque, 2007; Pascual 207 et al. 2012); to investigate a possible involvement of these receptors in the presynaptic 208 modulation of glutamatergic transmission upon TRPV1 activation, capsaicin was 209 applied with the mGluR5 antagonist MPEP (50μM) or with the non-selective group I 210 mGluR antagonist MCPG (100μM). In both assays capsaicin significantly increased 211 mEPSC frequency (n=7, p= 0.040 paired sample T-test and n=8, p=0.042 paired 212 sample T-test, respectively), suggesting that mGluR are not involved in the upstream 213 TRPV1 signalling. Both MPEP and MCPG did not induce changes in basal synaptic 214 properties. (Ctrl 7.62203 +/- 0.83 Hz, MPEP 7.95877+/- 1.10 Hz; Ctrl 19.16708 +/- 215 0.98 pA MPEP 18.83072 +/- 0.69 pA, n=7, p=0.61 and p=0.62 respectively, paired 216 sample T-test; Ctrl 6.19 +/-0.67606 MCPG 6.66+/- 0.71Hz; Ctrl 19.46 +/-0.97 217 MCPG 18.66 +/-1.18,n=8, p=0.10 and p=0.13, paired sample T-test). 218 219 220 221

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Supplementary Figure 13: Effects of ARN14988 and SB203580 on MV release 222 and neurotransmission 223

224 225 (a) Summary graph of mEPSC frequency (left) and amplitude (right) before (black 226 empty bar) and after 3 μM ARN 14988 (gray empty bar) (n=10, p=0.84 and *p<0.05, 227 Paired Sample t-test). (b) Microglia cell cultures were challenged with vehicle, caps 228 or ARN+caps and MVs shedding were measured. Histogram shows the fold increase 229 of total EV concentration detected by Nanosight upon stimulation of microglia 230 cultures with vehicle (ctrl, black bar), capsaicin (light gray bar) or ARN+capsaicin 231 (dark grey bar; One way Anova *p<0.05,**p<0.01 Dunn's Method for multiple 232 comparison test). (c) Changes of mEPSCs evoked by MVs previously treated with 233 ARN+caps (ctrl n=10, caps n=8, p<0.01 Mann-Whitney Rank Sum Test). (d) As in 234 (b) but with 400nM SB203580 (blue bar; One way Anova ****p<0.0001 followed by 235 Tukey’s multiple comparison test) 236 237 238 239 240 241 242 243

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(a) Example of binary (digital) silhouettes of microglia cells from cortical slices 247 acquired by transforming the tiff files of Iba1-immunoreactive cells to binary files by 248 means of the ImageJ software. The cell area values and the TI were calculated for 249 each silhouette and the obtained area/TI ratio value was used to classify each cell in a 250 specific category (resting, ipertrophied, bushy and amoeboid).(b) Plot of stochastic 251 sampling of microglia cells belong to a specific category from WT (black bars) and 252 TRPV1-/- (empty bar) cortical slices (n=5 for both WT and TRPV1-/-). Note that in no 253 treated-TRPV1-/- tissue there is a higher amount of hypertrophied microglia cells than 254 in WT. (c) Stack column graph showing a greater percentage of activated cells 255 (striped bar) related to resting microglia (filled bar) in WT slices (grey bars) treated 256 for 10 minutes with LPS (500ng/ml). The same treatment carried out on slices from 257 TRPV1-/- mice (blue bars) produced no changes in the number of activated cells. 258 (*p<0.05, **p<0.01 Fisher's exact test). 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280

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Supplementary Figure 15: Electrophysiological features of CCI PNs in baseline 281 conditions and after TRPV1 activation 282

283 284 (a) Comparison between average input-out (input current/number of spikes) curves 285 for the two conditions, shams (black dots) and CCI (red dots). The sciatic nerve 286 ligation caused a leftward shift of the input current to action potential curves, with a 287 significant increase of firing rate over a range of current injections. P<0.05 Mann-288 Whitney test. (b) Average input current/number of spike curves for control (red) and 289 after IRTX (green) from CCI PN recordings. The inhibition of TRPV1s significantly 290 reverted the leftward shift of the input-output curve induced by the surgery of the 291

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sciatic nerve (*p<0.05, **p<0.005, Paired Sample Wilcoxon Signed Rank test). (c) 292 On the left, cumulative probability curves of mEPSC interevent intervals for the total 293 recorded neurons from sham (n=16, black line) and CCI mice (n=14, red line) 294 (p<0.001 Kolmogorov-Smirnov test). Right, summary histograms and line series plots 295 of mEPSC from PNs of shams and CCI mice (6.47±0.58 and 8.63±0.92 Hz shams 296 [n=16] and CCI mice [n=14], respectively p<0.05 Two sample T-test). (d) Left, 297 cumulative probability distributions of mEPSC amplitudes of total recorded PNs from 298 shams (in black) and CCI (in red), p<0.001 KS test. Right, summary histograms and 299 line series plots of mEPSC amplitudes from PNs of sham and CCI mice (16.76±0.65 300 and 12.2±0.67 pA, respectively, p<0.01 Two sample T-test. (e) Cumulative 301 probability distributions of interevent intervals (left) amplitude (center) and area (left) 302 of mEPSCs recorded from 4 cells of CCI mice in the presence of minocycline (p<0.01, 303 KS test). (f) Voltage-clamp recording of layer 2/3 PN from a 4 week CCI mice in the 304 presence of picrotoxin, APV, TTX, and DNQX. Application of capsaicin induced an 305 inward current that was reverted by IRTX. (g) Summary plot of changes in IM after 306 application of capsaicin and subsequent application of IRTX (n=4, *p<0.05 307 **p<0.01). 308 309 310 311 312 Supplementary Figure 16: Development of allodynia after CCI. 313 314

315 Mechanical threshold (force-grams) was measured in naive (no CCI, n=10) and CCI 316 mice (n=11) at 3 (D3; DF1,19; F 83,02; p<0.0001), 7 (D7; DF 1,19; F 118,19; p<0.0001) 317 and 21 days (D21; DF1,19; F 28,05; p< 0.0001) after neuropathy induction. 318 319 320 321 322 323 324 325

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Supplementary Figure 17: Schematic summary of the proposed TRPV1 326 mechanism in the cortex of mice in physiological and neuropathic pain condition. 327 328

329 (a) In the cortex of healthy mice, TRPV1s are preferentially expressed in microglia. 330 Activation of these channels induces the microvesicles (MVs) shed from microglia 331 surface. Once released, these MVs may enhance spontaneous excitatory 332 neurotransmission by increasing the ready releasable pool of vesicles together with 333 the release probability. Additional players in the microglia to neuron communication 334 could be microglial factors released either from the breakdown of MVs or directly 335 from the microglia surface (such as IL-1β, NGF, ATP, NO, TNFα and glutamate). (b) 336 In mice suffering from neuropathic pain, TRPV1 are also localized in principal 337 cortical neurons, besides microglial cells. In this scenario, while microglial TRPV1 338 activation accounts for the increase of glutamate release, activation of postsynaptic 339 TRPV1 directly affects pyramidal neuron excitability by changing membrane 340 potential and likely increasing AMPA receptor trafficking. 341 342 343 344 345 346 347 348

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Supplementary Figure 18. 349

350 Full western blots from which the cropped images in Supplementary Figure 4. 351 352 SUPPLEMENTARY REFERENCES 353 354 1. Li, Y., Du, X. F., Liu, C. S., Wen, Z. L. & Du, J. L. Reciprocal regulation between 355 resting microglial dynamics and neuronal activity in vivo. Dev Cell 23, 1189-1202 356 (2012). 357 2. Ji, K., Akgul, G., Wollmuth, L. P. & Tsirka, S. E. Microglia actively regulate the 358 number of functional synapses. PLoS One 8, e56293 (2013). 359 3. Zhou, Q. et al. Decreased miR-199 augments visceral pain in patients with IBS 360 through translational upregulation of TRPV1. Gut 65, 797-805 (2016). 361

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4. Jiang, G. et al. Expression levels of microRNA-199 and hypoxia-inducible factor-1 362 alpha in brain tissue of patients with intractable epilepsy. Int J Neurosci 126, 326-334 363 (2016). 364 5. Luo, H., Cheng, J., Han, J. S. & Wan, Y. Change of vanilloid receptor 1 expression 365 in dorsal root ganglion and spinal dorsal horn during inflammatory nociception 366 induced by complete Freund’s adjuvant in rats. Neuroreport 15, 655-658 (2004). 367 6. McKelvey, R., Berta, T., Old, E., Ji, R. R. & Fitzgerald, M. Neuropathic pain is 368 constitutively suppressed in early life by anti-inflammatory neuroimmune regulation. 369 J Neurosci 35, 457-466 (2015). 370 7. Latremoliere, A. & Woolf, C. J. Central sensitization: a generator of pain 371 hypersensitivity by central neural plasticity. J Pain 10, 895-926 (2009). 372 8. Barrientos, R. M. et al. Peripheral infection and aging interact to impair 373 hippocampal memory consolidation. Neurobiol Aging 27, 723-732 (2006). 374 9. Konsman, J. P., Parnet, P. & Dantzer, R. Cytokine-induced sickness behaviour: 375 mechanisms and implications. Trends Neurosci 25, 154-159 (2002). 376 10. Chen, T. et al. Postsynaptic potentiation of corticospinal projecting neurons in the 377 anterior cingulate cortex after nerve injury. Mol Pain 10, 33 (2014). 378 11. Zhuo, M. Cortical excitation and chronic pain. Trends Neurosci 31, 199-207 379 (2008). 380 12. Bushnell, M. C., Ceko, M. & Low, L. A. Cognitive and emotional control of pain 381 and its disruption in chronic pain. Nat Rev Neurosci 14, 502-511 (2013). 382 13. Moreira, F. A., Aguiar, D. C., Terzian, A. L., Guimaraes, F. S. & Wotjak, C. T. 383 Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 384 channels in fear and anxiety-two sides of one coin? Neuroscience 204, 186-192 385 (2012). 386 14. Turkheimer, F. E. et al. The methodology of TSPO imaging with positron 387 emission tomography. Biochem Soc Trans 43, 586-592 (2015). 388 15. Marinelli, S. et al. Botulinum neurotoxin type A counteracts neuropathic pain and 389 facilitates functional recovery after peripheral nerve injury in animal models. 390 Neuroscience 171, 316-328 (2010). 391 16. Fujita, H. et al. Effects of GM-CSF and ordinary supplements on the ramification 392 of microglia in culture: a morphometrical study. Glia 18, 269-281 (1996). 393 17. Szabo, M. & Gulya, K. Development of the microglial phenotype in culture. 394 Neuroscience 241, 280-295 (2013). 395 18. Chiurchiù, V. et al. Proresolving lipid mediators resolvin D1, resolvin D2, and 396 maresin 1 are critical in modulating T cell responses. Sci Transl Med 8, 353ra111 397 (2016). 398 19. Harris, J. A. et al. Anatomical characterization of Cre driver mice for neural 399 circuit mapping and manipulation. Front Neural Circuits 8, 76 (2014). 400

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20. Ringkamp M, et al. Capsaicin responses in heat-sensitive and heat-insensitive A-401 fiber nociceptors. J Neurosci. 21, 12 (2001). 402 403 404 405 406