Matilde Cordero-Erausquin

Dhanasak Dhanasobhon

Maria-Carmen Medrano

Léa J Becker

Yunuen Moreno-Lopez

Sehrazat Kavraal

Charlotte Bichara

Rémy Schlichter

Perrine Inquimbert

Ipek Yalcin

Enhanced analgesic cholinergic tone in the spinal cord in a mouse model of neuropathic pain

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Endogenous acetylcholine (ACh) is an important modulator of nociceptive sensory processing in the spinal cord. Epidural administration of neostigmine, an acetylcholinesterase (AChE) inhibitor that induces an increase in ACh spinal level by preventing its degradation, produces pain relief for child birth and post-operation pain in clinics [START_REF] Eisenach | Epidural neostigmine: will it replace lipid soluble opioids for postoperative and labor analgesia?[END_REF]. In rodents, local spinal injection of AChE inhibitors (by intrathecal injections, or i.t.) similarly produces analgesia to thermal or chemical noxious stimulation [START_REF] Chen | Spinal GABAB receptors mediate antinociceptive actions of cholinergic agents in normal and diabetic rats[END_REF][START_REF] Chiari | Sex differences in cholinergic analgesia I: a supplemental nicotinic mechanism in normal females[END_REF][START_REF] Hartvig | Cholinergic mechanisms in pain and analgesia[END_REF][START_REF] Miranda | Neostigmine interactions with non steroidal anti-inflammatory drugs[END_REF][START_REF] Naguib | Antinociceptive effects of spinal cholinesterase inhibition and isobolographic analysis of the interaction with mu and alpha 2 receptor systems[END_REF]. The analgesic effect of endogenous ACh is not only observed when its level is artificially increased (by AChE antagonists), but also with its physiological levels. Indeed, impairment of cholinergic signaling by locally antagonizing ACh receptors (nicotinic or muscarinic) or by knocking-down the β2 subunit of nAChRs induces hyperalgesia and/or allodynia in rodents [START_REF] Hama | Antagonist of nicotinic acetylcholine receptors (nAChR) enhances formalin-induced nociception in rats: tonic role of nAChRs in the control of pain following injury[END_REF][START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF]Yalcin et al., 2011b;[START_REF] Zhuo | Tonic cholinergic inhibition of spinal mechanical transmission[END_REF]. This suggests that a basal "tone" of spinal ACh modulates the nociceptive threshold.

A dense plexus of cholinergic processes (composed of both dendrites and axons) lies in laminae (L) II-III of the dorsal horn (DH) of rodents [START_REF] Barber | The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study[END_REF][START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. Within this plexus, cholinergic dendrites receive synaptic contacts from primary afferents, and cholinergic axons make presynaptic contacts onto them [START_REF] Olave | Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn[END_REF][START_REF] Ribeiro-Da-Silva | Substantia Gelatinosa of the Spinal Cord[END_REF], offering a potential substrate for sensory modulation. In rodents, sparse cholinergic neurons with extended ramification, are located in lamina III-IV and have been proposed to be the main source of this plexus. They could therefore be responsible for cholinergic analgesia [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. Interestingly, we have demonstrated that a similar cholinergic population exists in the dorsal horn of macaque monkeys, with comparable localization, density and most importantly, synaptology [START_REF] Pawlowski | A novel population of cholinergic neurons in the macaque spinal dorsal horn of potential clinical relevance for pain therapy[END_REF]. The behavioral and anatomical similarities have confirmed that mice are a valuable model to study cholinergic analgesia. But how this population of cholinergic interneurons ultimately impacts nociceptive behavior is still unknown.

AChE inhibitors are used in the clinics only in a context of acute pain. Yet chronic pain is also a devastating and widespread problem, inadequately treated according to two-thirds of chronic pain sufferers [START_REF] Breivik | Survey of chronic pain in Europe: prevalence, impact on daily life, and treatment[END_REF]. How the endogenous spinal cholinergic control of analgesia evolves in situations of chronic pain in primate is unknown, and data on rodents are scarce and at times contradictory. While Rashid and Ueda have proposed that the cholinergic tone is disrupted in neuropathic animals as shown by the loss of effect of the i.t. injection of a nicotinic antagonist [START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF], i.t. injections of AChE antagonists still have an analgesic effect [START_REF] Hwang | The antiallodynic effects of intrathecal cholinesterase inhibitors in a rat model of neuropathic pain[END_REF][START_REF] Lavand'homme | Intrathecal neostigmine, but not sympathectomy, relieves mechanical allodynia in a rat model of neuropathic pain[END_REF][START_REF] Lavand'homme | Sex differences in cholinergic analgesia II: differing mechanisms in two models of allodynia[END_REF][START_REF] Takasu | Spinal alpha(2)-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury[END_REF] suggesting that ACh is still locally available to affect sensory processing. The dorsal horn of the spinal cord is a site for numerous forms of plasticity occurring during chronic pain [START_REF] Tsuda | Neuronal and microglial mechanisms for neuropathic pain in the spinal dorsal horn and anterior cingulate cortex[END_REF], and it was recently demonstrated that synaptic inputs or intrinsic properties of several interneuronal populations are altered during the neuropathy [START_REF] Cheng | Identification of spinal circuits involved in touch-evoked dynamic mechanical pain[END_REF][START_REF] Imlach | Glycinergic dysfunction in a subpopulation of dorsal horn interneurons in a rat model of neuropathic pain[END_REF][START_REF] Peirs | Dorsal Horn Circuits for Persistent Mechanical Pain[END_REF][START_REF] Petitjean | Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury[END_REF]. How the cholinergic population, and more generally the spinal cholinergic analgesia, is affected in chronic pain models remained to be evaluated and was the main goal of this study.

Accordingly, we have combined behavioral, histological, in vivo and in vitro electrophysiological recordings to improve our understanding of spinal cholinergic analgesia. We specifically addressed the question of its plasticity in conditions of chronic pain, and of potential underlying mechanisms.

Material and methods

Animals

This study utilized male wild-type and transgenic CD1 and C57BL/6 mice between 3 -10 weeks. Two different transgenic lines have been used: ChAT::EGFP CD1 [START_REF] Von Engelhardt | Functional characterization of intrinsic cholinergic interneurons in the cortex[END_REF] and β2*-nAChR knock-out (KO) C57BL/6J mice [START_REF] Picciotto | Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain[END_REF]. The animals were group-housed between two to six animals per cage and maintained on a 12-hour light/dark cycles with food and water provided ad libitum. The animal facility Chronobiotron UMS3415 is registered for animal experimentation under the Animal House Agreement B6748225. All protocols were approved by the "Comité d'Ethique en Matière d'Expérimentation Animale de Strasbourg" (CREMEAS, CEEA35) and the Ministère de l'enseignement supérieur, de la recherche et de l'innovation under the reference 02729.01.

Cuff surgery and behavioral assessment

The cuff model is used to induce neuropathic pain in mice [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF]. The surgery was carried out under ketamine (1 µl/g-Imalgene1000, Merial), azepromazin (0,60 µl/g -Calmivet, Vetoquinol) and medetomidine (1,18 µl/g -Domitor, Orion pharma/Elanco) anesthesia. The surgery was performed as previously described [START_REF] Yalcin | The sciatic nerve cuffing model of neuropathic pain in mice[END_REF]. Briefly, a 2 mm section of split PE-20 polyethylene tubing (Harvard apparatus, Les Ulis, France) was placed around the right common branch of sciatic nerve in the cuff mice group. Sham-operated mice underwent the same surgical procedure without cuff implantation. After performing the surgery, Atipamezol chlorhydrate (Antisedan, Orion pharma/Vetoquinol) mix (10 µl/g of animal), an antidote to medetomidine, was provided to speed up the recovery from the anesthesia. After a one-week recovery period, the neuropathic phenotype was assessed with von Frey tests.

We assessed the role of spinal cholinergic modulation on mechanical threshold in naïve and neuropathic CD1 mice with von Frey filaments [START_REF] Yalcin | Chronic treatment with agonists of beta(2)-adrenergic receptors in neuropathic pain[END_REF]. Experimental groups were designed to ensure comparable number of animals in sham and cuff groups. Prior to the surgery, the animals were divided into groups based on mechanical threshold to ensure the average in each group was similar. For each experimental session (several sessions were required to perform a full doseresponse curve), at least a pair of sham and cuff mice received saline 0.9% injection while the others received the drug of interest. Intrathecal (i.t.) injections were performed under gaseous anesthesia (3% Isoflurane for induction and 2% for maintenance) as previously described (Yalcin et al., 2011b). Mice were habituated for 15 minutes in clear Plexiglas boxes (7cm x 9 cm x 7 cm) on an elevated mesh screen. Filaments of increasing diameter were slowly brought to the plantar surface of each hind paw and removed after a slight bent. The mechanical threshold is determined as the first filament inducing at least three withdrawals out of five consecutive trials. The results were expressed in grams. For naïve animals, the left and right paw data were averaged for each time point. The mice were tested prior to injection or surgery to establish the baselines. A minimum of three tests were performed over 3 to 7 days to determine the mechanical withdrawal threshold. Motor coordination was tested using a rotarod (Bioseb). Mice were trained on 2 consecutive days where they ran at constant speed of 8 and 16 rotations per minute (rpm) without falling for at least 60 secs. On the experimental day, the rod was programmed to accelerate from 0 to 40 rpm over 300 s. Each mouse was allowed three trials where the average rpm and time at the point of failure was recorded to establish the baseline. Subsequently, mecamylamine was intrathecally injected and the mice were tested again 3 times starting after 15 min post-injection.

In vitro electrophysiological recordings

In vitro patch-clamp recordings were performed from ChAT::EGFP CD1 mice, 7 to 24 days postsurgery (sham or cuff); the investigator was not blind to the group. The mice were anesthetized with a mix of i.p. ketamine (200mg/kg -Imalgene 1000, Merial) and xylazine (20 mg/kg -Rompun 2%, Bayer). The animals were transcardially perfused for approximately 3 minutes with ice-cold sucrosebased Artificial Cerebral Spinal Fluid (ACSF) composed of (in mM): 252 Sucrose, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 Glucose, 26 NaHCO3, 1.25 NaH2PO4, 2 kynurenic acid, continuously bubbled with carbogen (95% O2/ 5% CO2). Two approaches were used to extract the spinal cord: hydraulic extrusion [START_REF] Chery | Junctional versus extrajunctional glycine and GABA(A) receptormediated IPSCs in identified lamina I neurons of the adult rat spinal cord[END_REF] or laminectomy [START_REF] Flynn | A horizontal slice preparation for examining the functional connectivity of dorsal column fibres in mouse spinal cord[END_REF]. A 200 to 300 µm horizontal slice (HS) was performed, containing the first four laminae of the DH. We also performed 350 to 400 µm thick transverse slices (TS). The slices were allowed a one-hour recovery period prior to recording at room temperature in oxygenated ACSF containing (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 Glucose, 26 NaHCO3, 1.25 NaH2PO4.

Cholinergic neurons were identified by the presence of the enhanced green fluorescent protein (EGFP) produced under the control of the cholineacetyltransferase (ChAT) promoter. Cells were recorded in the lumbar cord, in the area corresponding to termination of the sciatic nerve (end of L3 -L6 spinal segments). In HS, LIII/IV was identified by few ChAT::EGFP neurons located on the surface of the first slice (positioned upside-down). In TS, LIII/IV was identified as the laminae just ventral to the translucent substantia gelatinosa (LII), or ventral to the EGFP+ cholinergic plexus [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. For voltage clamp experiments, a cesium (Cs) based intracellular solution was used (in mM): 80 Cs2SO4, 5 KCl, 2 MgCl2, 10 HEPES, 10 Biocytin (pH = 7.3). The cells were maintained at -60 mV and 0 mV for excitatory and inhibitory post-synaptic currents respectively. For current clamp recordings, a K-based intracellular solution was used (in mM): 136 CH3KO3S, 2 MgCl2, 10 HEPES, 10 Biocytin. For both intracellular solutions, the liquid junction potential was not corrected. The recordings were made through the Axopatch 200A (Axon instruments) or the Multiclamp 700A (Molecular devices). The current traces were filtered at 5 kHz before digitalization via a digitizer (BNC-2110, National Instruments) at 20 kHz. Data acquisition was performed using WinEDR and WinWCP softwares (Strathclyde Electrophysiology Software, John Dempster, University of Strathclyde, Glasgow, UK).

For voltage clamp, the synaptic events (recorded over a 5-minute period) were detected with the threshold search method with WinEDR software. The threshold was set, for miniature and spontaneous EPSCs, to absolute amplitudes ≥ 2 pA and a duration ≥ 1.25 ms. For miniature and spontaneous IPSCs, the thresholds were set to absolute amplitudes ≥ 3 pA and a duration ≥ 1.75 ms. A baseline track-time of 5 ms was implemented prior to each event. Following the automatic detection by the software, all captured events were inspected individually, and only events that possessed a quick rise followed by an exponential decay were kept. The absolute frequency is calculated as the number of events over the total duration of the recording. The peak amplitudes were measured for accepted events via semi-automated procedures in WinWCP. However, individual events were excluded if they contained overlapping events or had an unstable baseline. The frequency and amplitude of currents were analyzed over 5-minute duration. For current clamp, the firing patterns were classified as previously reported [START_REF] Prescott | Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat[END_REF].

In vivo electrophysiological recordings

In vivo electrophysiological recordings were performed from ChAT::EGFP CD1 mice, 8 to 21 days post-surgery (sham or cuff); the investigator was not blind to the group. The mice were anesthetized with urethane (2.5 g/ kg, i.p.) and the trachea was cannulated. Rectal temperature was continuously monitored, and the animal was maintained at 35.5°C using a heating pad (TC-1000; Bioseb). The mouse was placed in a stereotaxic and spinal frame with 2 clamps fixed on its vertebra to immobilize the vertebral column. A laminectomy was performed to expose L3-L5 segments of the spinal cord, and a small chamber (approximately 0.1 ml) was created with 2% agar around the exposed lumbar spinal cord. After removal of the dura, the spinal cord was covered with saline (NaCl 0.9%) or with drugs diluted in saline (see below).

Single-unit extracellular recordings from spinal dorsal horn neurons were performed as previously described [START_REF] Medrano | Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain[END_REF]. Only neurons responding to mechanical stimulation of the ipsilateral hind paw were included in this study. Recordings were made with a glass electrode (Harvard Apparatus, Holliston, MA) filled with 0.5M CH3COOK (resistance: 15-25 mV). A motorized micromanipulator (Narishige, Tokyo, Japan) was used to gradually descend the electrode with 4-mm steps until the single-unit activity of a neuron was recorded. The recording electrode was inserted at depths ≤500 mm from the surface of the spinal cord (corresponding to lamina I to V). The signal was amplified (IR-183; Cygnus Technology, Delaware Water Gap, PA), filtered at 0.3 to 3 kHz (Brownlee amplifier; AutoMate Scientific, Berkeley, CA), and digitized at 20 kHz with a MICRO3-1401 (CED, Cambridge, United Kingdom). Data were analyzed offline with Spike 2 software (CED). The cutaneous receptive field of the recorded neuron was identified by touching the ipsilateral hind paw.

The response to a non-nociceptive mechanical stimulus, touch, and to a nociceptive one, pinch, was determined as described before and expressed as the mean frequency (in Hz) of the neuronal firing rate during the 10 seconds that lasted the peripheral stimulation [START_REF] Medrano | Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain[END_REF]. The touch stimulus was applied by brushing the skin with a camel's hair brush for 10 seconds (8-10 times). The pinch stimulus was applied by means of small serrated forceps (Graefe forceps; Fine Scientific Tools, Vancouver, Canada) for 10 seconds.

Mecamylamine hydrochloride (nicotinic receptor antagonist; Sigma (St. Louis, MO, USA)) was applied directly at the surface of the dorsal horn using the agar chamber described above. Stock solutions were first prepared in water at 100x their final concentration, stored at -20 °C and, on the day of the experiment, diluted in saline to their final concentration. The drug was applied topically onto the recording site of the spinal cord after careful removal of saline of the pool. Ten minutes following drug application, the response of neurons to the mechanical stimuli was tested [START_REF] Medrano | Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain[END_REF]. All solutions were applied at 34-35°C.

Tissue fixing and staining

ChAT::EGFP mice were transcardially perfused under pentobarbital (Ceva Sante Animal, 54.7mg/ml, i.p. injection) with 0.1M phosphate buffer (PB, pH 7.4) followed by 4% Paraformaldehyde (PFA) in PB for 15 minutes. The lumbar part of the spinal cord was sliced in 40μm-thick transverse sections using a vibrating blade microtome (VT 1000S, Leica, Rueil-Malmaison, France), and serially collected in wells. The sections were washed three times with PBS and saturated with PBS/ 0.5% Triton X-100/ 5% donkey serum at room temperature for 45 minutes. The sections were incubated at room temperature overnight in PBS/ 0.5% Triton X-100/ 1% donkey serum with a goat polyclonal ChAT antibody (1:500 dilution, Chemicon Millipore, AB 144P). The goat polyclonal anti-ChAT antibody stains a single band of 68-70 kDa apparent molecular weight on Western blot analysis of mouse brain lysate (manufacturer's technical information). The specificity of this antibody for the antigen has been determined by preadsorption with the appropriate purified protein as described by others [START_REF] Rico | A population of cholinergic neurons is present in the macaque monkey thalamus[END_REF], indicating that after preadsorption there was no staining. The staining pattern that we obtained was identical to that used in previous studies in the spinal dorsal horn by us and others [START_REF] Barber | The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study[END_REF][START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF][START_REF] Olave | Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn[END_REF][START_REF] Pawlowski | A novel population of cholinergic neurons in the macaque spinal dorsal horn of potential clinical relevance for pain therapy[END_REF]. After three PBS washes, the slides were incubated with a CY3 anti-goat antibody (1:400 dilution, Jackson Immunoresearch) for 2 hr. The sections were finally washed three times with PBS and mounted with fluorescent mounting media (Dako, Les Ulis, France). Slides were examined under fluorescence using a microscope (Leica) and the 30 sections centered on the lumbar 4 / lumbar 5 border were identified [START_REF] Watson | Atlas of the Mouse Spinal Cord[END_REF]. From these 30 sections, we performed a systematic sampling by considering every second section (i.e. 15x 40µm-thick sections, located at 80 μm intervals, centered at L4/L5): on both the contralateral and ipsilateral DH, we counted the number of EGFP+ neurons (indicating the expression of the ChAT::EGFP transgene), the number of CY3+ neurons (indicating the binding of the anti-ChAT antibody), and checked whether these two fluorophores colocalized (the very low density of cholinergic neurons allows colocalization studies with an epifluorescence microscope) .

Tissue harvesting and RT-qPCR

Two weeks after cuff surgery, and after control of their mechanical threshold through Von Frey measures, sham and cuff animals were anaesthetized by isoflurane inhalation and killed by decapitation. Spinal cord was extracted by hydraulic extrusion and right and left lumbar section were dissected and RNA extraction was performed immediately after using the Qiagen RNeasy Mini Kit (Hilden Germany) according to manufacturer instruction. Data were normalized to gapdh and B2M (from the same animal) and fold changes were calculated using the 2-ΔΔCt method [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF]. Specifically, the ∆Ct for each probe was normalized to the average ∆Ct of the same probe in sham samples present in the same qPCR plaque.

Statistical analysis

The statistical analysis was performed with the statistical software R (version 3.4.1) in conjunction with Graphpad software (Prism 7 for Mac, GraphPad Software, Inc., San Diego, CA, USA).

Behavior data (time-course): The von Frey data is a discrete ordinal data, and it did not pass the conditions to use parametric tests. For more than 2 time points, we thus analyzed it using the nonparametric test "nparLD", provided as a plug-in by the statistical software R [START_REF] Noguchi | nparLD: An R Software Package for the Nonparametric Analysis of Longitudinal Data in Factorial Experiments[END_REF].

nparLD provides an ANOVA type multiple-factor analysis taking into account a longitudinal variable (time) along with multiple dependent (right vs left paw) and independent (surgery; drug treatment) variables. A post-hoc test (Bonferroni test) was performed only when a main effect was observed.

For the β2 KO mice, we used Kruskal-Wallis test followed by Dunn's multiple comparison. The rotarod experiments were statistically assessed using unpaired t-test.

Behavior date (dose-response): As baseline mechanical thresholds were different for sham and cuff animals, dose-response curves were built using the "relative response" calculated as:

= 100 * VF(15min) VF(0min)
, where VF is the mechanical threshold (Von Frey) at the indicated time for each drug concentration.

As saline is theoretically a "zero" concentration of the test drug that cannot be represented in the log dose-response curve, we attributed it to a 0.0001 nmol drug value. For the mecamylamine dataset, a non-linear regression we performed to fit the dose response curve. The fit allowed the determination of an IC50 for each group. For the physostigmine dataset, its was best fitted by a linear regression that did not allow determination of an EC50. For comparison between the groups, we used the extra sumof-squares f test to determine if one fit (no difference between groups) or two fits (statistical difference between groups) would best fit the data.

In vitro electrophysiology data: Frequency, amplitude and most cell properties were analyzed with the In vivo electrophysiology data: Statistical evaluation was carried out with repeated measures 2-way ANOVA (Surgery, Drug).

In addition, the depths for in vivo recordings were compared with unpaired t-test (effect of surgery), and the number of labelled cell (histology data) with a 2-way ANOVA (Surgery, Contralateral vs.

Ipsilateral).

qPCR: For each probe and each side (contralateral, ipsilateral), the FC values for sham and cuff were compared through non-paired t-test.

The significance level was set at P <0.05 and data were expressed as mean ± SEM for graphs.

Results

Behavioral effect of cholinergic modulation onto mechanical responses

We first characterized the dose-dependent effect of spinal cholinergic modulation onto transmission of mechanical information in naïve CD1 mice. For this purpose, the mechanical paw withdrawal threshold (PWT) was evaluated with the von Frey test, before and after i.t. injection of the AChE inhibitor physostigmine. Physostigmine (15 nmol, i.t), produced a long-lasting increase in the mechanical withdrawal threshold while lower doses (1.5-7.5 nmol) were without effect (Fig. 1A; statistics are presented in the figure legends, and, when needed, first order comparisons are illustrated in the Supplementary figures; here Suppl. Fig. 1). This indicates that ACh is endogenously released and its level in the spinal cord closely controlled by AChE. We thus confirm that, if its concentration is increased (by inhibiting its hydrolysis), ACh reaches downstream targets, activating a spinal circuit leading to analgesia.

In order to investigate whether this cholinergic analgesic mechanism is active in baseline physiological conditions, we interfered with it by blocking cholinergic nicotinic receptors. We thus injected the non-specific nicotinic antagonist mecamylamine (10-400 nmol, i.t.) and tested at different time points after the injection (Fig. 1B). Mecamylamine induced a dose-and time-dependent reduction in the PWT: at all doses tested, the PWT was statistically reduced as soon as 15 min post-injection, and for all but the 10 nmol dose, the effect lasted for more than two hours. In the 15 min -2 hours time window, there was no significant difference between the three higher doses (at each time point, 400 nmol or 100 nmol vs. 20 nmol : p>0.99, Bonferroni's multiple comparisons test) suggesting that the effect already plateaued with 20 nmol mecamylamine. 150 minutes after the injection, however, animal that had received 20 nmol of mecamylamine had a PWT statistically different from the one of animals that had received 100 or 400 nmol mecamylamine (20 nmol vs. 100 nmol, p=0.0007 ; 20 nmol vs. 400 nmol, p=0.0001, Bonferroni's multiple comparisons test). Indeed, the allodynia evoked by 10 nmol mecamylamine had faded away after 150 minutes, while it was still present in the 20, 100 and 400 nmol injection groups (comparisons with saline presented in Fig. 1B). This suggests that the cholinergic analgesic mechanism is active in baseline physiological conditions, either because ACh is continuously released and activating downstream circuits, or because it is released as a consequence of the mechanical stimulation. Whatever the underlying cause, the pharmacological effect of a nicotinic antagonist defines the presence of an analgesic spinal cholinergic tone, acting via nicotinic receptors.

High concentrations of mecamylamine are known to have potentially non-specific effects [START_REF] Mcdonough | A study of the N-methyl-D-aspartate antagonistic properties of anticholinergic drugs[END_REF][START_REF] O'dell | Mecamylamine is a selective non-competitive antagonist of Nmethyl-D-aspartate-and aspartate-induced currents in horizontal cells dissociated from the catfish retina[END_REF]. We used β2 nicotinic knock-out mice (β2 KO) to confirm that the highest dose of mecamylamine used (400 nmol) was acting specifically through inhibition of nAChR receptors (Suppl. Fig. 2A illustrates that 400 nmol mecamylamine induces a marked reduction of the PWT in WT animals, but not on β2 KO mice ). Moreover, we observed that this high concentration of mecamylamine did not affect motor-coordination as evaluated by the rotarod test in naive mice (Suppl. Fig. 2B illustrates the absence of effect of 400 nmol mecamylamine on the rotarod test), suggesting that the observed modulation strictly involved mechanical sensory processing.

The spinal cholinergic tone is present after neuropathy

We then investigated the alteration of this analgesic cholinergic tone in a mouse model of neuropathy.

We used the cuff model and analyzed the effect of i.t. injection of mecamylamine (10-400 nmol) on the PWT of sham and cuff mice. For the ipsilateral, but not the contralateral, paw there was a significant interaction between Surgery, Time and Drug (p = 0.0354), indicating that the neuropathy modified the response of the ipsilateral paw to mecamylamine.

Mecamylamine induced a dose-dependent reduction in the mechanical withdrawal threshold of the contralateral hindpaw in sham and cuff mice ( Bonferroni's multiple comparisons test). The mechanical allodynia observed in cuff animals was exacerbated when higher doses were injected (Fig. 2A; post-hoc tests at 15 min, in cuff mice: for 100 nmol mecamylamine vs. saline, p = 0.0017 and for 400 nmol mecamylamine vs. saline, p<0.0001;

Bonferroni's multiple comparisons test). Analysis of the dose-response curve demonstrates a shift to the right in cuff mice, with higher doses being necessary to obtain a significant effect (Fig. 2B;

Ipsilateral side -Extra sum-of-squares f test, p<0.001). The IC50 was significantly shifted from 6.554 to 27.78 nmol after neuropathy on the ipsilateral side. Thus the spinal cholinergic tone is still present in cuff animals, involving nAChRs and affecting mechanical responses. The shift to the right of the mecamylamine dose-response curves further suggests that the cholinergic tone might be increased by the pathology, either because more ACh is released or because endogenous ACh has an increased effect on its post-synaptic targets. We next investigated these two possibilities.

Plasticity of LIII/IV cholinergic interneurons during neuropathy

Lamina III-IV cholinergic neurons are the main source of the spinal cholinergic tone [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. We thus closely analyzed this population in sham and neuropathic mice to test the hypothesis that the release of ACh could be increased following the neuropathy. In particular, we assessed 3 different criteria: (1) synaptic inputs, (2) active and passive electrophysiological properties, and (3) number of cholinergic interneurons.

Synaptic inputs to lamina III-IV interneurons

We then assessed whether DH cholinergic neurons could be differentially activated in sham vs. cuff by comparing the synaptic inputs received by these neurons in these two conditions. Cholinergic interneurons are located in lamina III-IV, and best preserved in horizontal slices [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. In such slices, we recorded spontaneous EPSCs and IPSCs from ChAT::EGFP and non-EGFP (unidentified) neurons in their close vicinity in sham and cuff mice (Fig. 3A). For EPSCs, there were no significant differences between the groups. For IPSCs, there was on average more inhibition in cuff vs. sham mice (not specific to any neuronal population under study), and cholinergic neurons had on average less inhibitory inputs than non-cholinergic ones (whether in sham or in cuff mice) (see Suppl.

Fig. 3). In contrast, as reported in other neuropathic models, we observed a decrease in the frequency of miniature IPSCs in lamina II (Suppl. Fig. 4). There was therefore no change of inputs frequency that was specific to cholinergic neurons after neuropathy (Fig. 3B).

These recordings reveal an increase in the frequency of inhibitory inputs that is not specific to cholinergic neurons as it is also observed in non-EGFP neurons in the same laminae If anything, this would argue that cholinergic neurons are less prone to firing (and releasing ACh) in neuropathic conditions. In order to test this hypothesis, we next compared the capacity of cholinergic neurons to be excited by direct depolarization in sham vs. cuff mice.

Active and passive electrophysiological properties

We analyzed the firing patterns of recorded neurons after injection of depolarizing currents, and classified their discharge profiles as either "tonic", "single-spike", or "phasic" (cf. Methods, Fig. 4A1). There was no statistical difference between ChAT::EGFP and non-EGFP neurons concerning the proportion of the different firing patterns in the two different animal groups (Fig. 4B). A fraction of recorded neurons presented spontaneous ongoing firing at rest (rheobase of 0pA, Fig. 4C) but their proportion was not different between types of neurons (ChAT::EGFP vs. non-EGFP) or type of surgery (sham vs. cuff) (3x3x3 contingency table, p = 0.2674). Specifically, 2 out of 6 ChAT::EGFP neurons (33.3%) were spontaneously active in sham mice, but none out of 8 (0%) in cuff, while 1 out of 7 non-ChAT neurons (14.2%) was spontaneously active in sham mice vs. 2 out 6 (33.3%) in cuff mice. There also was no statistical difference in the rheobase in the different groups (Fig. 4C). We compared the input-output properties of the different neurons (measured as mean instantaneous firing frequency induced by an increasing depolarizing current, Fig. 4D). On average, there was no effect of the surgery on the instantaneous firing frequencies, but there was an effect of the intensity of the injected current, as well as of the type of neurons: ChAT::EGFP vs. non-EGFP neurons (see Suppl.

Fig. 5). Moreover, there was an interaction between the response to different injected currents and the type of neurons, as non-EGFP neurons fired more than ChAT::EGFP neurons when large currents were injected (120 to 180 pA). There was no interaction of these factors with the surgery, suggesting that the input-output properties of the neurons were not affected by the surgery. We also investigated the passive membrane properties of recorded neurons. There was no difference among groups (ChAT::EGFP or non-EGFP neurons, sham or cuff) in the resting potential (Fig. 4C), the input resistance (Fig. 4C) or the amplitude of a sag current (Fig. 4A2,4E).

Altogether, these measures indicate that the network plasticity observed in slices obtained from cuff mice does not alter the excitability of cholinergic neurons.

Number of cholinergic interneurons

There is an on-going debate on whether there is a loss of neurons in the DH that would contribute to the development of hyperalgesia or allodynia following neuropathy. Therefore, we investigated whether DH cholinergic interneurons were affected. We have previously shown that ChAT::EGFP mice are a good model for studying DH cholinergic neurons [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF]. Here, we took every second section from L4 -L5 spinal segments into account, and confirmed that all lamina III-IV EGFP+ are ChAT immunoreactive (n=62 neurons in 2 sham mice and n=56 neurons in 2 cuff mice); similarly, we could observe EGFP fluorescence in the vast majority of DH ChAT immunoreactive neurons (62 out of 63 neurons in 2 sham mice and 56 out of 56 neurons in 2 cuff mice). We compared the number of EGFP+ neurons in the contralateral and ipsilateral DH of adult ChAT::EGFP+ mice two weeks after surgery (Fig. 5). On the ipsilateral dorsal horn, there was no statistical difference between the number of ChAT::EGFP+ neurons per 40 µm thick sections in sham (on average 2.13 ± 0.38 neurons) vs. cuff (2.07± 0.38 neurons). This ruled out a change in the number of DH cholinergic neurons after neuropathy.

Changes in the downstream network responding to the cholinergic tone

As we found no evidence of specific increased excitation or excitability of DH cholinergic neurons in neuropathic animals, nor a change in neuronal density, we next asked whether the increased impact of the cholinergic tone in these animals could be explained by modifications in direct or indirect targets of cholinergic neurons, i.e. downstream of the tone itself. The cholinergic tone has never yet been described in in vivo electrophysiological recordings, nor its consequences on the response of DH neurons to tactile stimulation. We thus first studied these aspects by recording DH neurons in vivo in sham and cuff animals to identify a potential substrate to the antinociceptive cholinergic tone and its plasticity, and then turned to pharmacological behavioral experiments.

A neuronal substrate for the antinociceptive cholinergic tone

We performed in vivo electrophysiological recordings of lumbar DH neurons in sham and neuropathic animals within the second and third week after surgery. Neurons were selected for their localization in the DH (Fig. 6B) and their response to mechanical stimulation of the hindpaw, either non-nociceptive (touch) and/or nociceptive (pinch) (Fig. 6A; see methods and [START_REF] Medrano | Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain[END_REF]). Local application of 100 µM mecamylamine (on the spinal cord) induced an increased response to mechanical stimulation in neurons recorded from both sham and cuff mice (Fig. 6C). The recorded neurons spanned many DH laminae (Fig. 6B) and the response amplitudes showed high variability,

precluding the identification of a potential differential effect of mecamylamine in sham vs. cuff mice.

These recordings however demonstrate for the first time in vivo the presence of a spinal cholinergic tone, acting through nicotinic receptors, attenuating mechanical responses. This could be a substrate for the antinociceptive spinal cholinergic tone observed in behavioral experiments.

Effect of an AChE inhibitor in neuropathic animals

In order to evaluate a potential change of the network downstream of the cholinergic tone, we turned back to behavioral experiments and compared the effect of endogenous spinal ACh in sham and cuff mice by i.t. injection an AChE inhibitor. Physostigmine effect on the contralateral paw was not affected by the surgery (Fig. 7A). For the ipsilateral paw in contrast, the surgery modified the response to physostigmine. Indeed, in neuropathic mice, 7.5 nmol physostigmine injected i.t. was able to alleviate the mechanical allodynia of the ipsilateral paw while it was ineffective to increase PWT in sham animals (Fig. 7A, at 15 min: 7.5 nmol physostigmine vs. saline p<0.0001 in cuff and p>0.99 in sham; Bonferroni's multiple comparisons test). The 15 nmol dose however was analgesic for both paws in both sham and cuff mice (Fig. 7A, 15 min: drug vs. saline p<0.0001 for both sham and cuff;

Bonferroni's multiple comparisons test). The fitted regression lines demonstrates that physostigmine differentially affected the relative mechanical response in the ipsilateral (but not contralateral) paw of cuff vs. sham (Fig. 7B, Ipsilateral side: cuff vs. sham p<0.001; Extra sum-of-squares f test). This favors the hypothesis of a post-synaptic plasticity affecting the downstream players of cholinergic transmission (i.e. post-synaptic receptors).

Changes in the expression of nicotinic receptor subunits after neuropathy

While the increased ACh level induced by physostigmine could act on both nicotinic and muscarinic cholinergic receptors, our previous experiments using mecamylamine suggests a plasticity occurring (at least) at the nicotinic transmission level. We thus analyzed the level of expression of various nAChR subunits (α2, α4, α6, α7, β2 and β4) in the spinal cord by real-time qPCR (RT-qPCR). RT-qPCR were carried out 2 weeks after neuropathic pain induction on sham (n=4) and cuff (n=5) animals. Ipsilateral and contralateral sides of the lumbar spinal cord were processed independently. In the contralateral side, no differences in mRNA level of the analyzed nAChR subunits were detected in sham vs. cuff animals. On the ipsilateral side, no differences between sham and cuff animals were observed in the mRNA levels of α2, α4, α6, α7, and β4, but there was a decrease in the β2 subunit mRNA level in cuff mice (Fig. 8, β2 subunit FC: sham v cuff p = 0.0295; unpaired t-test).

All together, these results suggest a plasticity of the cholinergic transmission following neuropathic pain induction. This plasticity apparently mostly relies on an alteration of β2 subunit of the nAChR.

Discussion

In this study, we performed an in depth analysis of the antinociceptive spinal cholinergic tone. We demonstrated the persistence and even increased impact of this tone in a model of neuropathic pain, and ruled out changes in lamina III-IV cholinergic neurons as a source of this plasticity. Instead, our data show an increase in the sensitivity of downstream targets to spinal ACh. Low doses of AChE inhibitors, without any effect in control mice, are analgesics in cuff mice which may open new avenues for therapeutical relief of neuropathic pain conditions.

The existence of a "spinal cholinergic tone" has been proposed in the early 1990's and confirmed since then [START_REF] Rashid | Tonic inhibitory role of alpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmission in the spinal cord in mice[END_REF][START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF][START_REF] Zhuo | Tonic cholinergic inhibition of spinal mechanical transmission[END_REF] to describe an endogenous mechanism whose presence is revealed by a pharmacological effect of cholinergic antagonists. Although the term is reminiscent of the word "tonic", it gives no inference on the way ACh is released: either repetitively (through the "tonic firing" of a cholinergic neuron), or phasically (upon a specific stimulation, e.g. nociceptive stimulation [START_REF] Eisenach | Cerebrospinal fluid norepinephrine and acetylcholine concentrations during acute pain[END_REF]). We here similarly define the "cholinergic tone" through the allodynic effect of i.t. mecamylamine injection, suggesting that in basal conditions ACh has an ongoing (or provoked) analgesic effect.

The cuff, a neuropathic pain model chosen for this study, was originally developed for rats [START_REF] Mosconi | Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: ultrastructural morphometric analysis of axonal alterations[END_REF] and then adapted for mice [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF]. In a previous study, we characterized by in vivo recordings the loss of inhibitory tone in the spinal DH in this model [START_REF] Medrano | Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain[END_REF]. We provide here for the first time in vitro recordings in the spinal cord of these mice and observed an overall increase in the frequency of inhibitory currents in lamina III-IV after neuropathy (see below). We also performed recordings in lamina II to compare our observations with those obtained in other neuropathic pain models. We observed a reduction in the frequency, but not amplitude, of miniature IPSC currents in lamina II similar to that reported in the spared nerve injury (SNI) and chronic constriction injury (CCI) models [START_REF] Iura | Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor delta subunit expression after chronic constriction injury of the sciatic nerve in mice[END_REF][START_REF] Moore | Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord[END_REF]. However, we observed no change in the frequency for mEPSCs while a reduction is reported in a study focusing on GABAergic interneurons in the CCI model [START_REF] Leitner | Impaired excitatory drive to spinal GABAergic neurons of neuropathic mice[END_REF]. It remains to be clarified whether this difference is due to models used, or to the fact that the the study of Leitner et al. focuses only on GABAergic neurons. Importantly, all of our experiments were conducted from the first up to the third week after cuff surgery, a time window where the allodynia phenotype is robustly established but where anxiodepressive-like behaviors are not yet present [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF]Yalcin et al., 2011a).

A previous study exploring the plasticity of the spinal cholinergic tone used the partial ligation of the sciatic nerve model, and analyzed the thermal paw withdrawal [START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF]. They showed that i.t. injection of 10 nmol mecamylamine had no effect in the injured mice, but higher doses were not tested. In the von Frey mechanical test, we similarly found that 10 nmol mecamylamine was without effect, but testing higher doses demonstrated that they exacerbated the allodynia, demonstrating a shift to the right of the dose-response curve of the antagonist. Therefore, rather than a loss of cholinergic tone, our results suggest that its impact is actually increased by the neuropathy.

We also investigated the possible sources of this plasticity. Our previous studies concluded that spinal lamina III-IV cholinergic neurons were the main source of ACh at this level and that their morphology and projections were well suited to impact nociceptive processing, both in rodents and in primates [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF][START_REF] Pawlowski | A novel population of cholinergic neurons in the macaque spinal dorsal horn of potential clinical relevance for pain therapy[END_REF]. These cholinergic interneurons are also GABAergic [START_REF] Mesnage | Morphological and functional characterization of cholinergic interneurons of the dorsal horn of the mouse spinal cord[END_REF][START_REF] Miles | Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion[END_REF], and the existence of GABAergic cell death after peripheral neuropathy and its contribution to mechanical allodynia has been proposed although controversially debated [START_REF] Inquimbert | NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain after Peripheral Nerve Injury[END_REF][START_REF] Polgar | Lack of evidence for significant neuronal loss in laminae I-III of the spinal dorsal horn of the rat in the chronic constriction injury model[END_REF][START_REF] Scholz | Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury[END_REF]. In the present study, we found no difference in the number of ChAT::EGFP neurons following neuropathy. Other morphological alterations could exist, such as a reduction in the number of appositions onto postsynaptic targets, as demonstrated for parvalbumin (PV) interneurons [START_REF] Petitjean | Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury[END_REF]. The decreased innervation of PKC-gamma neurons by PV neurons induces a decreased feed-forward inhibition that 'gates' the transmission of non-noxious information into nociceptive circuits, and is proposed to be a substrate of mechanical allodynia [START_REF] Petitjean | Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury[END_REF]. Numerous studies illustrate the synaptic plasticity occurring in this feed-forward network [START_REF] Abraira | The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn[END_REF][START_REF] Alba-Delgado | Subpopulations of PKCgamma interneurons within the medullary dorsal horn revealed by electrophysiologic and morphologic approach[END_REF][START_REF] Cheng | Identification of spinal circuits involved in touch-evoked dynamic mechanical pain[END_REF][START_REF] Foster | Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch[END_REF][START_REF] Imlach | Glycinergic dysfunction in a subpopulation of dorsal horn interneurons in a rat model of neuropathic pain[END_REF][START_REF] Lu | A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia[END_REF][START_REF] Peirs | Dorsal Horn Circuits for Persistent Mechanical Pain[END_REF], but the position of cholinergic neurons with respect to this network is yet unknown [START_REF] Cordero-Erausquin | Neuronal networks and nociceptive processing in the dorsal horn of the spinal cord[END_REF]. We observed an increased frequency of inhibitory currents in laminae III-IV but this does not translate into decreased excitability, indicating that the release of ACh might not be significantly impaired. Membrane properties of these neurons were unchanged in neuropathic animals,

similarly to what has been reported for LIII (GAD67 or not) or LII GABAergic neurons [START_REF] Gassner | Properties of spinal lamina III GABAergic neurons in naive and in neuropathic mice[END_REF][START_REF] Schoffnegger | Physiological properties of spinal lamina II GABAergic neurons in mice following peripheral nerve injury[END_REF]. Thus changes in membrane excitability or altered firing patterns in the spinal cord dorsal horn are unlikely causes for the alterations underlying neuropathic pain or for the observed plasticity of the cholinergic tone.

We then explored downstream mechanisms, i.e. cholinoresponsive elements of the spinal nociceptive network. Rat cervical projection neurons were known to have an increased response to mechanical stimulation after topical application of an AChE inhibitor [START_REF] Chen | Activation of muscarinic receptors inhibits spinal dorsal horn projection neurons: role of GABAB receptors[END_REF]. Using similar in vivo recordings in mice, but on unidentified DH neurons, our study illustrates that a nicotinic antagonist has the opposite effect, demonstrating for the first time a potential substrate of the analgesic cholinergic tone in vivo. Our recordings also show that this tone is still present after cuff injury, but reveal no alteration after neuropathy. Interestingly, both the response to a non-nociceptive (touch) and nociceptive (pinch) stimuli were similarly affected by the nicotinic antagonist, suggesting that the cholinergic tone controls both types of afferents or downstream neurons, possibly common to the different mechanical modalities.

To finally identify the plasticity that could explain the increased impact of the cholinergic tone in cuff mice, we analyzed the analgesic properties of increasing i.t. doses of an AChE inhibitor, physostigmine, with the von Frey test. These experiments demonstrated a shift to the left of the dose response curve of this drug since doses that were subthreshold in sham mice returned the PWT in the cuff to pre-surgery levels. This is reminiscent of data obtained in the rat after systemic (i.p.) injection of another AChE antagonist in the spared nerve injury model [START_REF] Kimura | Relief of hypersensitivity after nerve injury from systemic donepezil involves spinal cholinergic and gamma-aminobutyric acid mechanisms[END_REF]. Similar observations of subthreshold (in naïve animals) intrathecal doses of AChE antagonist being antihyperalgesic after SNL are reported for the thermal sensory modality in mice [START_REF] Chiari | Sex differences in cholinergic analgesia I: a supplemental nicotinic mechanism in normal females[END_REF][START_REF] Naguib | Antinociceptive effects of spinal cholinesterase inhibition and isobolographic analysis of the interaction with mu and alpha 2 receptor systems[END_REF][START_REF] Takasu | Spinal alpha(2)-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury[END_REF]. Interestingly, nicotinic agonists also have analgesic properties after i.t. injection in the PNL mouse model, both in mechanical and thermal tests, at doses (10 nmol nicotine, 0.3 nmol epibatidine) inefficient in sham mice [START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF]. This, together with our results, strongly suggests that major changes occur at the receptor level, either in density or in composition. The proteins forming the nAChR have proven difficult to study with immunohistochemical approaches due to lack of specificity of antibodies [START_REF] Moser | Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures[END_REF]. Most studies, including ours, thus rely on an analysis of mRNA levels although we acknowledge that it only partially informs on the plasticity that can take place, because of potential post-traductional that could alter the stability of proteins. We have shown a reduced level of β2 mRNA in the ipsilateral lumbar cord of cuff mice, while α2, α4, α6, α7 and β4 mRNA levels were unchanged. While this result demonstrates a plasticity occurring at the post-synaptic level, it does not directly explain an increased efficacy of the endogenous cholinergic tone. β2 containing nAChRs are indeed the receptors with the highest affinity for ACh (especially when receptors have the (α4)2(β2)3 stochiometry, [START_REF] Nelson | Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors[END_REF]) so a reduction in the expression of β2 should favor receptors with lower affinity (such as the (α4)3(β2)2 stoichiometry one). On the other side, the reduced expression of β2 could contribute to the allodoynic phenotype of cuff mice, as we have previously shown that β2-knock out mice have a decreased mechanical threshold (Yalcin et al., 2011b). This is reminiscent of the negative correlation observed between the mRNA level of the α6 nAChR subunit (variable across inbred mice strains) and 20 the amount of allodynia the mice develop after spare-nerve injury [START_REF] Wieskopf | The nicotinic alpha6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors[END_REF]. In contrast with our results, a study in rats using a peripheral axotomy model found an increase in the mRNA levels for a5 and b2 in the axotomy dorsal horn vs. control [START_REF] Yang | Peripheral nerve injury induces trans-synaptic modification of channels, receptors and signal pathways in rat dorsal spinal cord[END_REF]. The study however does not distinguish between ipsi and contralateral alterations, and does not provide behavior experiments, thus preventing analysis of the cholinergic tone plasticity in this model and a direct comparison with our results.

In this study, we have focused on the spinal nicotinic tone because we have revealed the cholinergic tone through antagonization of nAChRs by mecamylamine. Other antagonists targeting more specifically α4β2 or α3β2 * /α6β2 * nAChRs [START_REF] Papke | Analysis of mecamylamine stereoisomers on human nicotinic receptor subtypes[END_REF] have been shown to have a similar effect in mice or rats [START_REF] Rashid | Tonic inhibitory role of alpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmission in the spinal cord in mice[END_REF][START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF]Yalcin et al., 2011b;[START_REF] Young | Spinal alpha3beta2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli[END_REF]. The fact that the allodynic effect of nicotinic antagonists is lost in nAChR β2 knock-out mice (this study and (Yalcin et al., 2011b)), and that the expression of this subunit is reduced in the ipsilateral lumbar spinal cord of cuff mice further confirms the involvement of β2-containing nAChRs.

In contrast, α7 * nAChRs do not seem to be involved in the cholinergic tone in mice [START_REF] Rashid | Tonic inhibitory role of alpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmission in the spinal cord in mice[END_REF][START_REF] Rashid | Neuropathy-specific analgesic action of intrathecal nicotinic agonists and its spinal GABA-mediated mechanism[END_REF]Yalcin et al., 2011b). However, it should be noted that the increased ACh level induced by AChE inhibition could act both on nicotinic and muscarinic post-synaptic receptors. In naïve rats, the analgesic effect of neostigmine on thermal responses has been shown to be mediated by muscarinic receptors in males but muscarinic and nicotinic receptors in females [START_REF] Chiari | Sex differences in cholinergic analgesia I: a supplemental nicotinic mechanism in normal females[END_REF][START_REF] Lavand'homme | Sex differences in cholinergic analgesia II: differing mechanisms in two models of allodynia[END_REF]. However muscarinic antagonists had no effect on von Frey responses when injected i.t. in naïve mice [START_REF] Honda | Intrathecal alpha2 adrenoceptor agonist clonidine inhibits mechanical transmission in mouse spinal cord via activation of muscarinic M1 receptors[END_REF][START_REF] Paqueron | Plasticity in action of intrathecal clonidine to mechanical but not thermal nociception after peripheral nerve injury[END_REF], suggesting that there might be species or sensory modality differences.

In this study, we have demonstrated that the spinal cholinergic tone modulating nociceptive responses was still present, and even more efficient, after peripheral nerve injury. Our data do not support a change in the number or electrophysiological properties of dorsal horn cholinergic interneurons, but rather favor a modification occurring at the post-synaptic site. Overall, the increased analgesic effect of i.t. AChE antagonists in neuropathic conditions is promising. Other endogenous analgesic systems, such as the conditioned pain modulation, appear to have a reduced efficiency in a variety of chronic pain conditions in humans [START_REF] Julien | Widespread pain in fibromyalgia is related to a deficit of endogenous pain inhibition[END_REF][START_REF] Yarnitsky | Conditioned pain modulation (the diffuse noxious inhibitory control-like effect): its relevance for acute and chronic pain states[END_REF]. In contrast, the increased analgesic efficiency of the spinal cholinergic tone suggests that this system can be successfully leveraged to relieve pain. Until now, AChE inhibitors have mainly been used in the clinics in situations of acute pain (parturition or post-operative [START_REF] Eisenach | Epidural neostigmine: will it replace lipid soluble opioids for postoperative and labor analgesia?[END_REF]). The fact that lower doses (thus with fewer side effects) could be efficient in chronic pain conditions opens new avenues for the treatment of neuropathic pain. The distribution of frequencies being non-normal, their log10 was taken to perform the ANOVA.

There was no statistical difference in EPSCs frequencies between groups (ChAT::EGFP+ vs. non-EGFP, Sham vs. Cuff). For IPSCs, main effects were significant: neuron (ChAT::EGFP vs. non-EGFP, p = 0.001378, 2-way ANOVA), surgery (sham vs. cuff, p = 0.046641, 2-way ANOVA).

  RNA concentration was measured by spectrophotometry (NanoDrop 1000, Thermo Fisher Scientific, Waltham, Massachusetts, United-States) and sample were then kept at -80°C for later use. To generate cDNA, 350 ng of the total RNA was reversed transcribed with M-MLV reverse transcriptase (Invitrogen, Carlsbad, California, United-States) in 40µL reaction containing oligo (dT) primers (Invitrogen, Carlsbad, California, United-States). Real-time quantitative polymerase chain reaction was performed in 10µl reaction containing 3µl of ten time's diluted cDNA, 5µl of PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, California, United-States), 1µl of a mix of forward and reverse primers (10 µM) and 1µl of H2O. The cycling conditions were 50°C for 2 min, 95°C for 10 min, and then 40 cycles at 95°C for 15 s and 60°C for 1 min. The sequences of the primers are: chrna2 : F: GTGCCCAACACTTCCGATG, R: TGTAGTCATTCCATTCCTGCTTT, chrna4: F: CGGCCA GTAGCCAATATCTC, R: CAGGTTGGTCGTCATCATCT, chrna6: F: CGGTTTATGTCTGTGGCTATG, R: CCCAACGCAATCTGTAGTCC, chrna7: F: ATCCCTGTAGCTGTCGGTCT, R: GCCGGTGATGGGTGTAAGAA, chrnb2: F: GTGGACGGTGTACGCTTCATTG, R: GGTCGTGGCAGTGTAGTTCTGG, chrnb4: F: TACAACAACCTGATCCGCCC, R: AGTCCATTCCTGTTTCAGCCA, b2m: F: TGCTACGTAACACAGTTCCACC, R: TCTGCAGGCGTATGTATCAGTC and gapdh : F: TGGCCTCCAAGGAGTAAGAAAC, R: TGGGATGGAAATTGTGAGGGAG.

  Fig. 2A Top, in sham, for all doses of mecamylamine vs. saline at 15 min, p<0.0001 in post-hoc tests; in cuff, similar comparison yielded to p<0.0001 for the 20 nmol, 100 nmol and 400 nmol mecamylamine doses, and p = 0.0257 for the 10 nmol dose, Bonferroni's multiple comparisons test). For the ipsilateral hindpaw, 10 and 20 nmol was ineffective in cuff as opposed to sham groups (Fig. 2A Bottom, post-hoc tests at 15 min, drug vs. saline: p = <0.0001 for 10 and 20 nmol mecamylamine in sham and respectively p>0.99 and p=0.159 in cuff;

Figure 1 :

 1 Figure 1: Time course of the effect of cholinergic drugs on mechanical threshold and motor coordination in naive animals.

Figure 2 :

 2 Figure 2: Mecamylamine shows that a spinal cholinergic tone is still present after neuropathy

Figure 3 :

 3 Figure 3: Number and synaptic inputs to Dorsal Horn cholinergic interneurons in sham and cuff mice

Figure 4 :

 4 Figure 4: Active and passive properties of laminae III/IV ChAT::EGFP+ and Non-EGFP cells in sham and cuff mice.

Figure 5 :

 5 Figure 5: The number of DH cholinergic interneurons is unaltered after cuff surgery

Figure 6 :

 6 Figure 6: Touch and pinch responses in spinal dorsal horn neurons are modulated by endogenous acetylcholine via nicotinic receptors.

Figure 7 :Figure 8 :

 78 Figure 7: Physostigmine unravels downstream changes to spinal cholinergic tone after neuropathy

  For post-hoc comparisons, interactions with only one or two variables were made with the Bonferroni test with multi-comparison correction. A more complex 3x3x3 contingency table was performed online at http://vassarstats.net (Log-Linear Analysis for an AxBxC Contingency Tablefoundunder Frequency data tab) for firing pattern, rebound spike observations and rheobase.

2-way ANOVA test. The original or transformed (log or inverse function) datasets were verified to show a normal distribution (Shapiro-Wilk normality test). The independent parameters analyzed were: Surgery, Neuron (ChAT::EGFP or non-EGFP), injected current, type of current (spontaneous or minis).

We gratefully acknowledge the support from the University of Strasbourg Institute for Advanced Study (USIAS) and ANR-13-JSV4-0003-01 GRANT to MCE. DD is the recipient of a Region Alsace doctoral fellowship, YML and LJB were supported by the Fondation pour la Recherche Médicale (respectively post-doctoral and doctoral fellowship), and SK by the NeuroTime Erasmus Mundus Joint Doctorate Neuroscience PhD program (funded by the European Commission), and CB a recipient of a fellowship from the French Ministry of Higher Education and Research. The authors express their gratitude towards J.L. Rodeau for his expert assistance on the statistical approaches, Philippe Isope and Yves De Koninck for critical readings; and Sophie Reibel-Foisset and the Chronobiotron (UMS 3415, Centre National de la Recherche Scientifique) for mice handling.