Mélanie Kremer

Salim Megat

Yohann Bohren

Xavier Wurtz

Laurent Nexon

Alice Rhian

Stéphane Ceredig

Dominique Doridot

Eric Massotte

Ipek Salvat

Yalcin

Rhian Alice Ceredig

Stéphane Doridot

Dominique Massotte

Eric Salvat

Ipek Yalcin

Michel Barrot
email: mbarrot@inci-cnrs.unistra.fr

Delta opioid receptors are essential to the

Keywords: β2-mimetics, terbutaline, formoterol, neuropathic pain, mechanical allodynia, opioid system, δ-opioid β2-mimetics, terbutaline, formoterol, neuropathic pain, mechanical allodynia, opioid system, δ-opioid AUC, area under the curve, β 2 -ARs, β 2 adrenoceptors, COPD, chronic obstructive pulmonary disease, DOP, delta opioid, i.p., intraperitoneal, KOP, kappa opioid, MOP, mu opioid, PWT, paw withdrawal threshold, SSNRIs, selective serotonin-noradrenaline reuptake inhibitors, SSRIs, selective serotonin reuptake inhibitors, TCAs, tricyclic antidepressant

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

Background

Antidepressant drugs including tricyclic antidepressants (TCAs) and selective serotonin-noradrenaline reuptake inhibitors (SSNRIs) are among the first-line treatments of neuropathic pain. [START_REF] Attal | EFNS guidelines on the pharmacological treatment of neuropathic pain: 2010 revision[END_REF][START_REF] Dworkin | Recommendations for the pharmacological management of neuropathic pain: an overview and literature update[END_REF][START_REF] Saarto | Antidepressants for neuropathic pain: a Cochrane review[END_REF][START_REF] Kremer | Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insights[END_REF] These drugs mainly act by blocking the transporters of noradrenaline and serotonin. The critical role of the noradrenergic component in neuropathic pain relief was first suggested based on the poor clinical effectiveness of selective serotonin reuptake inhibitors (SSRIs). [START_REF] Attal | EFNS guidelines on the pharmacological treatment of neuropathic pain: 2010 revision[END_REF][START_REF] Dworkin | Recommendations for the pharmacological management of neuropathic pain: an overview and literature update[END_REF][START_REF] Saarto | Antidepressants for neuropathic pain: a Cochrane review[END_REF][START_REF] Max | Effects of desipramine, amitriptyline, and fluoxetine on pain in diabetic neuropathy[END_REF] The lack of SSRIs action can also be observed preclinically in rodent models of neuropathic pain. [START_REF] Kremer | Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insights[END_REF][START_REF] Benbouzid | Chronic, but not acute, tricyclic antidepressant treatment alleviates neuropathic allodynia after sciatic nerve cuffing in mice[END_REF] This raised the question of the potential role of the adrenoceptor(s), downstream of noradrenaline, in the action of the antidepressant drugs. We previously showed, by using a peripheral nerve injury model, [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF] that the antiallodynic effect of chronic antidepressant drugs is mediated by β 2 adrenoceptors (β 2 -ARs), [START_REF] Bohren | Antidepressants suppress neuropathic pain by a peripheral beta2-adrenoceptor mediated anti-TNFalpha mechanism[END_REF][START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Yalcin | Beta2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain[END_REF][START_REF] Yalcin | beta(2)-adrenoceptors are critical for antidepressant treatment of neuropathic pain[END_REF] but not by α 2 , nor β 1 or β 3 -ARs. Following these findings, it has been suggested in animals that β 2 -ARs agonists could be beneficial in peripheral neuropathic pain conditions. [START_REF] Yalcin | Chronic treatment with agonists of beta(2)-adrenergic receptors in neuropathic pain[END_REF][START_REF] Choucair-Jaafar | Beta2-adrenoceptor agonists alleviate neuropathic allodynia in mice after chronic treatment[END_REF] These data were further supported by some clinical observations, showing in a case report that a β 2 -mimetic induced significant relief in 6 patients with neuropathic pain, [START_REF] Cok | Is there a place for beta-mimetics in clinical management of neuropathic pain? Salbutamol therapy in six cases[END_REF] and in a retrospective epidemiologic study that the relative incidence of postthoracotomy neuropathic pain was 5-fold lower for patients with a chronic β 2 -mimetic treatment. [START_REF] Salvat | Effects of beta2 agonists on post-thoracotomy pain incidence[END_REF] Interestingly, the antidepressant drug mechanism leading to relief of mechanical hypersensitivity after chronic treatment is also delta-opioid (DOP) receptor dependent. [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Bohren | Mu-opioid receptors are not necessary for nortriptyline treatment of neuropathic allodynia[END_REF][START_REF] Megat | kappa-Opioid receptors are not necessary for the antidepressant treatment of neuropathic pain[END_REF][START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF] The opioid system plays a leading role in inhibitory controls of pain. [START_REF] Mogil | Pain genes?: natural variation and transgenic mutants[END_REF] It is involved in the direct action of analgesics targeting mu-opioid (MOP) receptors [START_REF] Gaveriaux-Ruff | Opioid receptor genes inactivated in mice: the highlights[END_REF] such as morphine, but also in the indirect recruitment of opioid receptors in the action of antidepressant drugs against neuropathic pain. [START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Marchand | Evidence for a monoamine mediated, opioid-independent, antihyperalgesic effect of venlafaxine, a non-tricyclic antidepressant, in a neurogenic pain model in rats[END_REF][START_REF] Mico | Antidepressants and pain[END_REF] In a similar way to antidepressant drugs, it has been suggested that the effect of β 2 -mimetics on neuropathic mechanical allodynia may also involve the endogenous opioid system. Indeed, an acute administration of naltrindole, a DOP receptor antagonist, temporarily suppressed the benefit of chronic treatment with the very long-acting β 2 -mimetic clenbuterol in a model of sciatic nerve compression, [START_REF] Yalcin | Chronic treatment with agonists of beta(2)-adrenergic receptors in neuropathic pain[END_REF] and the effect of the β 2 -mimetic terbutaline in a model of diabetic neuropathy. [START_REF] Choucair-Jaafar | The antiallodynic action of nortriptyline and terbutaline is mediated by beta(2) adrenoceptors and delta opioid receptors in the ob/ob model of diabetic polyneuropathy[END_REF] These first data suggested that DOP receptors would be necessary for the therapeutic effect of β 2 -mimetics on neuropathic pain, but still required to be genetically confirmed as well as to test the role of the other opioid receptors. Based on their half-life, β 2 -mimetics can be classified as short-acting β 2 -mimetics, such as terbutaline, salbutamol, F o r P e e r R e v i e w 4 fenoterol and pirbuterol, which are prescribed for the curative treatment of asthma attacks and chronic obstructive pulmonary disease (COPD) or the threat of preterm labor; or as long-acting β 2 -mimetics, such as formoterol and salmeterol, which are used as bronchodilators to help controlling and preventing COPD symptoms. Using transgenic and pharmacological approaches, we evaluated here the role of the three opioid receptors in the antiallodynic action of two β 2 -mimetics, one with a short half-life, terbutaline (5-6h), and the other with a long half-life, formoterol (12h). [START_REF] Cazzola | beta2-agonist therapy in lung disease[END_REF] In the present study, independently of their half-life, both chronic terbutaline and formoterol reversed mechanical hypersensitivity in our model of neuropathic pain. Using genetic and pharmacological approaches, we also showed that DOP receptors, but neither MOP nor kappa-opioid (KOP) receptors, are necessary for this action of β 2 -mimetics.

Methods

Animals

Experiments were performed using C57BL/6J mice (Charles River, L'Arbresle, France) between 8 and 10 weeks-old at the time of surgery and in mice deficient for MOP, DOP or KOP receptors or β 2 -ARs and their littermate controls. The generation of mice lacking MOP, DOP or KOP receptors, or β 2 -ARs has been previously described. [START_REF] Filliol | Mice deficient for deltaand mu-opioid receptors exhibit opposing alterations of emotional responses[END_REF][START_REF] Matthes | Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene[END_REF][START_REF] Simonin | Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal[END_REF][START_REF] Chruscinski | Targeted disruption of the beta2 adrenergic receptor gene[END_REF] Heterozygote mice were bred in our animal facilities and genotyping of the litters was done upon weaning. Experiments using transgenic mice were conducted on adult male and female wild type and knockout littermate mice (for formoterol experiments, to refine the use of animals and test gender aspects) or on male only (for terbutaline experiments), weighing 20-30 g. When we used male and female, each experimental group contained the same number of males and females.

As the wild type animals have the same background and the same behavior, they were pooled to form the control groups. For other experiments with C57BL/6J mice, we used males only.

Model of neuropathic pain

Neuropathic pain was induced by cuffing the main branch of the right sciatic nerve. [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF][START_REF] Yalcin | The sciatic nerve cuffing model of neuropathic pain in mice[END_REF] Surgeries were performed under ketamine (68 mg/kg) / xylazine (10 mg/kg) intraperitoneal (i.p.) anesthesia (Centravet, Tadden, France). The common branch of the right sciatic nerve was exposed and a cuff of PE-20 polyethylene tubing (Harvard Apparatus, Les Ulis, France) of standardized length (2 mm) was unilaterally inserted around it (Cuff group). The shaved skin was closed using suture. Sham-operated mice underwent the same surgical procedure without implantation of the cuff (Sham group). All treatments started between two and three weeks after the surgery.

Measure of mechanical allodynia

Mechanical allodynia was tested using von Frey hairs and results were expressed in grams. Tests were done during the morning, starting at least 2 hours after lights on. Mice were placed in clear Plexiglas boxes (7 cm x 9 cm x 7 cm) on an elevated mesh screen. Calibrated von Frey filaments (Bioseb, Vitrolles, France) were applied to the plantar surface of each hindpaw until they just bent, in a series of ascending forces up to the mechanical threshold. Filaments were tested five times per paw and the paw withdrawal threshold (PWT) was defined as the lower of two consecutive filaments for which three or more withdrawals out of the five trials were observed. [START_REF] Yalcin | The sciatic nerve cuffing model of neuropathic pain in mice[END_REF][START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF] The person who conducted the tests was blinded to the treatments.

Treatment procedures

The terbutaline treatment began fifteen days after the neuropathy was induced, and it was maintained at least three weeks. During the treatment, mice received two intraperitoneal injections per day (morning and evening) of terbutaline hemisulfate (0.5 mg/kg, 5 mL/kg, Sigma-Aldrich, St. Quentin Fallavier).

The dose was chosen based on a previous dose-response experiment [START_REF] Choucair-Jaafar | Beta2-adrenoceptor agonists alleviate neuropathic allodynia in mice after chronic treatment[END_REF] . The drug was dissolved in 0.9 % NaCl solution that was also used for control injections. The injection of naltrindole (5 mg/kg, subcutaneous, Sigma-Aldrich, St. Quentin Fallavier), a DOP receptors antagonist, was done 36 days after surgery, i.e., after 21 days of terbutaline treatment, in wild-type, MOP and KOP receptors-deficient mice. The injection of naloxone methiodide (5 mg/kg, subcutaneous, Sigma-Aldrich, St. Quentin Fallavier), an opioid receptor antagonist that does not cross the blood-brain barrier, was done following the same procedure as for naltrindole, but in C57BL/6J mice. In order to evaluate the mechanical thresholds for nociceptive response, mice were submitted to von Frey tests before and 30 min after the antagonist injection.

Formoterol (Biotrend Chemicals AG, Zürich, Switzerland) was delivered per os through the drinking water with ad libitum access as sole source of fluid. The drug was dissolved in water with 0.02% saccharin to increase palatability and control mice received a solution of 0.02% saccharin in water (vehicle solution). To assess the daily dose of β 2 -mimetic that was actually received by the animals (in µg/kg/day), the bottles containing the treatment were regularly weighed and we calculated the ratio between the intake amount of formoterol per cage and the weight of the animals. A first set of experiments (Figure 3) included mice treated with oral formoterol at concentrations 0.5, 0.1, 0.05 or 0.001 µg/mL and their controls, and Sham mice that received the highest dose of formoterol (0.5 µg/mL) to test whether it had an analgesic effect per se (n = 5 in each group). Considering the volume of solution drank by the mice per 24 h (between 3.5 and 5 mL per mouse), the 0.5 µg/mL solution was equivalent to 7.27±0.48 µg/kg/day, the 0.1 µg/mL solution was equivalent to 1.78±0.10 µg/kg/day, the 0.05 µg/mL solution was equivalent to 0.98±0.05 µg/kg/day and the 0.001 µg/mL solution was equivalent to 0.2±0.01 µg/kg/day (Figure 3(c)).

Mice lacking MOP, DOP or KOP receptors received formoterol treatment (0.5 µg/mL), in the drinking water, following the same protocol. Data were pooled from four independent experiments, due to the non-regular production of mouse breeding. Four additional sets of mice were also used to pharmacologically assess the role of opioid receptors. Each set was composed of three groups, a sham and a cuff group treated with the oral vehicle, and a cuff group treated with formoterol (0.5 µg/mL). After three weeks of oral treatment, mice received a subcutaneous injection of the MOP receptor antagonist naloxonazine (Sigma-Aldrich, St. Quentin Fallavier, France, 30 mg/kg) or the DOR receptor antagonist naltrindole (5 mg/kg) or a saline solution (0.9 % NaCl). All drugs were dissolved in NaCl 0.9%. In order to evaluate the mechanical threshold, mice were submitted to von Frey testing before, 30 min and 1h after the antagonist injection.

Data and statistical analysis

Data are expressed as mean ± SEM. In figures 1(c) and 3(b), cumulated paw withdrawal thresholds during treatments were quantified as the area-under-the-curve (AUC) above the pre-treatment value, calculated by the trapezoidal method. [START_REF] Celerier | Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine[END_REF] Statistical analyses were performed with STATISTICA 10 (Statsoft, Tulsa, OK, USA) using multifactor analysis of variance (ANOVA). The surgery procedure (Sham or Cuff) and the treatments were taken as between-group factors. When needed, the time of measurement (either time course or preinjection vs. postinjection data) was taken as a within-subject factor. The Duncan test was used for post hoc comparisons. The significance level was set at p < 0.05.

Results

Terbutaline treatment in opioid receptor deficient mice

Cuff-implantation induced an ipsilateral mechanical hypersensitivity in wild-type mice, as previously described [START_REF] Benbouzid | Sciatic nerve cuffing in mice: a model of sustained neuropathic pain[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Bohren | Mu-opioid receptors are not necessary for nortriptyline treatment of neuropathic allodynia[END_REF][START_REF] Megat | kappa-Opioid receptors are not necessary for the antidepressant treatment of neuropathic pain[END_REF][START_REF] Yalcin | The sciatic nerve cuffing model of neuropathic pain in mice[END_REF] (Figure 1(a); F 7,231 = 10.25, p < 0.001; detailed statistics for all results are given in Table 1 in Supplemental Material). As previously showed [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Bohren | Mu-opioid receptors are not necessary for nortriptyline treatment of neuropathic allodynia[END_REF][START_REF] Megat | kappa-Opioid receptors are not necessary for the antidepressant treatment of neuropathic pain[END_REF][START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF] , this induction and maintenance of mechanical hypersensitivity was also similar along the experiment between wild-type, MOP -/-, DOP -/- and KOP -/-mice (Figure 1(b)) (even though enhanced allodynia between DOP -/-and their wild-type controls has been reported by others in the partial ligation model). [START_REF] Nadal | Neuropathic pain is enhanced in deltaopioid receptor knockout mice[END_REF] Two weeks after Cuff insertion, we started the treatments with terbutaline (0.5 mg/kg) or the control saline solution (0.9 % NaCl), twice a day. Terbutaline treatment alleviated the cuff-induced allodynia in wild-type mice after about 13 days To confirm this critical role of DOP receptors, we then tested the consequences of an acute injection of naltrindole in the wild-type, MOP and KOP receptor-deficient mice of the above experiment.

After 3 weeks of treatment with terbutaline or saline, an acute injection of naltrindole induced a relapse of mechanical hypersensitivity in these mice (Figure 2(a); WT: F 1,33 = 18.83, p < 0.001; MOP -/-: F 1,21 = 21.61, p < 0.001; KOP -/-: F 1,20 = 5.05, p < 0.01). We also controlled that naltrindole per se had no effect in mice with Sham surgery, or in neuropathic mice treated with saline.

Peripherals opioid receptors are involved in terbutaline antiallodynic effect

The acute injection of naloxone methiodide (5 mg/kg), an opioid receptor antagonist that does not cross the blood-brain barrier, induced an acute relapse of allodynia in Cuff-implanted mice chronically treated with terbutaline (Figure 2(b); F 1,20 = 10.9, p < 0.01). The same dose of naloxone methiodide had no effect in mice with Sham surgery treated with saline.

Antiallodynic action of chronic oral formoterol: dose response

For treatment with the long-acting β 2 -mimetic formoterol, we chose to use chronic oral administration, which first required determining the appropriate dose (Figure 3). Cuff implantation induced an ipsilateral mechanical allodynia (Figure 3(a); F 11,242 = 5.8, p < 0.001). We did not observe any change in the nociceptive threshold on the left paw or in the Sham group. Nineteen days after surgery, we started treatments with formoterol (0.001, 0.05, 0.1 or 0.5 µg/mL) or with saccharin vehicle solution (0.02% saccharin). Chronic formoterol treatment at doses 0.05, 0.1 and 0.5 µg/mL alleviated the cuff-induced allodynia after nineteen days, fifteen days and thirteen days of treatment respectively (Figure 3 h, the 0.001 µg/mL solution was equivalent to 0.2±0.01 µg/kg/day, the 0.05 µg/mL solution was equivalent to 0.98±0.05 µg/kg/day, the 0.1 µg/mL solution was equivalent to 1.78±0.10 µg/kg/day and the 0.5 µg/mL solution was equivalent to 7.27±0.48 µg/kg/day (Figure 3(c)). These amounts were in fact mostly taken over the 12 h night period, period during which mice usually drink.

To test whether formoterol had an analgesic effect per se, we tested the higher dose (0.5 µg/mL) in Sham control mice. This chronic oral treatment did not affect mechanical thresholds of the Sham group (Figure 3(d)).

Chronic oral formoterol treatment in opioid and β 2 -adrenergic receptor deficient mice

As previously observed, [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Kremer | The antiallodynic action of pregabalin in neuropathic pain is independent from the opioid system[END_REF] mechanical sensitivity thresholds of female mice were significantly lower than in males (baseline thresholds: 3.4 g ± 0.7 for females, 4.9 g ± 0.9 for males, t 11 = 4.18, p < 0.005).

Both male and female mice developed mechanical allodynia after surgery, and formoterol treatment suppressed the cuff-induced allodynia in both sexes (Figure 4 MOP-, DOP-, KOP-receptors or β 2 -AR deficient mice displayed the same baseline for mechanical sensitivity and cuff-induced mechanical hypersensitivity as their wild-type littermates, as previously observed. [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Yalcin | Beta2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain[END_REF][START_REF] Yalcin | beta(2)-adrenoceptors are critical for antidepressant treatment of neuropathic pain[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Bohren | Mu-opioid receptors are not necessary for nortriptyline treatment of neuropathic allodynia[END_REF][START_REF] Megat | kappa-Opioid receptors are not necessary for the antidepressant treatment of neuropathic pain[END_REF] Formoterol treatment alleviated cuff-induced allodynia in wild-type mice (Figure 4 To confirm the role of DOP receptors, independently from any developmental or lasting alteration of nociceptive pathway that may accompany opioid receptor deletion, we tested the consequence of an acute injection of the MOP receptor antagonist naloxonazine and the DOP receptor antagonist naltrindole (Figure 5). After 3 weeks of treatment with formoterol or saccharin, an acute injection of naltrindole temporarily suppressed the antiallodynic effect of formoterol treatment (Figure 5; F 2,24 = 5.3, p < 0.005); while naloxonazine had no effect on mechanical thresholds for paw withdrawal. We also observed that naltrindole had no effect per se in mice with Sham surgery or in mice that received saccharin alone.

Effect of opioid receptor antagonists on long lasting formoterol treatment

Discussion

In the present work, we assessed the role of the opioid receptors in the antiallodynic effect of two β 2mimetics, one with a short half-life, terbutaline, given intraperitoneally, and the other one with a long half-life, formoterol, given orally. We found that DOP receptors, but not MOP or KOP receptors, were required to the effect of those two β 2 -mimetics on mechanical hypersensitivity in a murine model of neuropathic pain.

Regardless of their half-life, terbutaline and formoterol exerted their effect with a similar therapeutic delay of about 13 days, which is also similar to the therapeutic delay of treatments with antidepressant drugs in the same model. [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Yalcin | Beta2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain[END_REF][START_REF] Yalcin | beta(2)-adrenoceptors are critical for antidepressant treatment of neuropathic pain[END_REF] Present data with oral formoterol and previous reports with sustained systemic injections of β-mimetics [START_REF] Yalcin | Chronic treatment with agonists of beta(2)-adrenergic receptors in neuropathic pain[END_REF][START_REF] Choucair-Jaafar | Beta2-adrenoceptor agonists alleviate neuropathic allodynia in mice after chronic treatment[END_REF] also suggest that the dose, once effective, has only a minor influence on this delay. The delay of action of these treatments in the context of neuropathic pain thus appears partly independent from the pharmacokinetic properties of the molecules and ways of administration, supporting the hypothesis that the delay may be more likely linked to the setting up of molecular and cellular neuroplasticity phenomena, as it has been described with antidepressant drugs in the context of depression. [START_REF] Duman | Pathophysiology of depression: the concept of synaptic plasticity[END_REF][START_REF] Vialou | Epigenetic mechanisms of depression and antidepressant action[END_REF][START_REF] Rantamaki | Antidepressant drug action--From rapid changes on network function to network rewiring[END_REF] β 2 -mimetics remained effective in mice deficient for MOP or KOP receptors, but lost their efficacy in mice deficient for DOP receptors, which seemed the sole opioid receptors necessary to the antiallodynic effect of β 2 -mimetics. This finding is further supported by pharmacological data. Indeed, the acute injection of the DOP receptor antagonist naltrindole led to a transitory relapse of mechanical allodynia in mice chronically treated with β 2 -mimetics. These results suggest a mechanism similar to the one previously observed for antidepressant drugs, whose antiallodynic action has also been shown to be dependent upon DOP receptors. [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF][START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF] In the cuff model it was also shown that sustained neuropathic pain condition was associated with a reduction of DOP receptor expression in small calcitonin gene-related peptide positive primary sensory neurons and in free nerve endings, that was not present in duloxetine [START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF] treated animals and that was partly corrected in formoterol [START_REF] Ceredig | Peripheral Delta Opioid Receptors Mediate Formoterol Anti-allodynic Effect in a Mouse Model of Neuropathic Pain[END_REF] treated animals. In physiological conditions, DOP receptor agonists have little noticeable effect on nociception, as shown by pharmacological studies and data in DOP receptor deficient mice. [START_REF] Gaveriaux-Ruff | Delta opioid receptor analgesia: recent contributions from pharmacology and molecular approaches[END_REF] However, under neuropathic pain conditions, the administration of DOP receptor agonists has been shown to decrease mechanical and 12 thermal pain modalities. [START_REF] Kabli | Anti-allodynic effects of peripheral delta opioid receptors in neuropathic pain[END_REF][START_REF] Hervera | The role of nitric oxide in the local antiallodynic and antihyperalgesic effects and expression of delta-opioid and cannabinoid-2 receptors during neuropathic pain in mice[END_REF][START_REF] Mika | The role of delta-opioid receptor subtypes in neuropathic pain[END_REF][START_REF] Saitoh | Delta Opioid Receptor (DOR) Ligands and Pharmacology: Development of Indolo-and Quinolinomorphinan Derivatives Based on the Message-Address Concept[END_REF][START_REF] Castany | The antinociceptive effects of a delta-opioid receptor agonist in mice with painful diabetic neuropathy: Involvement of heme oxygenase 1[END_REF] It has thus been suggested that DOP agonists may be of interest for pain treatment. Unfortunately, agonists of the DOP receptors cannot be presently used as appropriate clinical treatments for neuropathic pain. Indeed, while DOP agonists would have a lower abuse potential and life-threatening risk than MOP receptor agonists, [START_REF] Gaveriaux-Ruff | Delta opioid receptor analgesia: recent contributions from pharmacology and molecular approaches[END_REF][START_REF] Gallantine | A comparison of the antinociceptive and adverse effects of the mu-opioid agonist morphine and the delta-opioid agonist SNC80[END_REF] the molecules presently available have adverse effects, [START_REF] Peppin | Delta opioid agonists: a concise update on potential therapeutic applications[END_REF] in particular convulsions or sleep apnea, that prevent their clinical use. β 2 -AR and DOP receptors are both necessary to the antiallodynic effect of antidepressant drugs [START_REF] Yalcin | Beta2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain[END_REF][START_REF] Yalcin | beta(2)-adrenoceptors are critical for antidepressant treatment of neuropathic pain[END_REF][START_REF] Benbouzid | Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia[END_REF] and of β 2 -mimetics. Interestingly, the contribution of these two receptors to an antinociceptive action has also been reported following intraplantar administration of a β-AR agonist in inflammatory pain, [START_REF] Binder | Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue[END_REF] as well as in the antiallodynic action of a prolonged treatment with curcuma in a model of sciatic nerve constriction. [START_REF] Zhao | Curcumin exerts antinociceptive effects in a mouse model of neuropathic pain: descending monoamine system and opioid receptors are differentially involved[END_REF] The mechanistic link between these two receptors is however still to be explored. Since acute administration of naloxone methiodide, an antagonist which does not pass the blood-brain barrier, suppressed the antiallodynic effect of chronic terbutaline treatment, the peripheral opioid system can thus be considered as critical. Again, this peripheral DOP receptor component has also been observed concerning the antiallodynic action of prolonged treatment with antidepressant drugs. [START_REF] Kremer | A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline[END_REF][START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF] DOP receptors are present on primary afferents [START_REF] Binder | Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue[END_REF][START_REF] Mennicken | Phylogenetic changes in the expression of delta opioid receptors in spinal cord and dorsal root ganglia[END_REF][START_REF] Busch-Dienstfertig | Opioid receptors and opioid peptide-producing leukocytes in inflammatory pain--basic and therapeutic aspects[END_REF] and expressed by several classes of dorsal root ganglia neurons. [START_REF] Ceredig | Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain[END_REF][START_REF] Francois | A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins[END_REF] On the other hand, the β2-ARs, whose mRNA levels may increase in the dorsal root ganglia following sciatic nerve transection [START_REF] Maruo | Modulation of P2X receptors via adrenergic pathways in rat dorsal root ganglion neurons after sciatic nerve injury[END_REF] but not after sciatic nerve cuffing, [START_REF] Bohren | Antidepressants suppress neuropathic pain by a peripheral beta2-adrenoceptor mediated anti-TNFalpha mechanism[END_REF] were proposed to be preferentially expressed by the glial satellite cells of dorsal root ganglia. [START_REF] Bohren | Antidepressants suppress neuropathic pain by a peripheral beta2-adrenoceptor mediated anti-TNFalpha mechanism[END_REF] These non-neuronal cells are the peripheral analogues of astrocytes of the central nervous system and are known to express β2-AR at their membrane in various species including humans. [START_REF] Trimmer | Combination of immunocytochemistry and radioligand receptor assay to identify beta-adrenergic receptor subtypes on astroglia in vitro[END_REF][START_REF] Mantyh | Beta 2-adrenergic receptors are expressed by glia in vivo in the normal and injured central nervous system in the rat, rabbit, and human[END_REF] Even though, in transfected cell cultures, the existence of heteromeric complexes of DOP receptors and β 2 -AR has been studied, [START_REF] Cao | The composition of the beta-2 adrenergic receptor oligomer affects its membrane trafficking after ligand-induced endocytosis[END_REF][START_REF] Jordan | Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation[END_REF][START_REF] Ramsay | Homo-and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences[END_REF] the above anatomical evidence suggests that both receptors would unlikely be coexpressed by the same cells in dorsal root ganglia. The immediate relapse after an acute administration of naltrindole rather suggests that the opioid component is likely located downstream of the adrenergic component. We may thus hypothesize that the recruitment of β 2 -AR could lead to an activation of the cAMP/PKA pathway in satellite glial cells, which could lead to synthesis and release of opioid peptides like enkephalins that would then stimulate DOP receptors present on primary afferents. [START_REF] Binder | Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue[END_REF][START_REF] Busch-Dienstfertig | Opioid receptors and opioid peptide-producing leukocytes in inflammatory pain--basic and therapeutic aspects[END_REF] Alternately, it could also be hypothesized that DOP receptors are simply part of a permissive mechanism, in which 13 their presence would be essential to the normal functioning of pathways responsible for the antiallodynic effect, without being per se an element of the molecular therapeutic cascade itself. This hypothesis could be supported by the fact that DOP receptors were shown to be more particularly involved in mechanical nociceptive responses. [START_REF] Scherrer | Dissociation of the opioid receptor mechanisms that control mechanical and heat pain[END_REF]

Conclusion

In summary, the present findings show that β 2 -mimetics, either intraperitoneal or per os, can have an antiallodynic action in a rodent model of sciatic nerve compression, and that DOP receptors are essential to it. Our results also show that neither MOP nor KOP receptors are necessary to this action of β 2mimetics. Our data are also supportive of a peripheral location of the opioid component of β 2 -mimetic effect. Together, these findings show a mechanistic convergence between β 2 -mimetics the known antidepressant drugs' action in neuropathic pain models. These findings also support the need of future research to elucidate the mechanistic link between β 2 -ARs and DOP receptors. Between 15 and 19 days post-surgery, the oral treatment with formoterol (0.5 µg/ml) or its saccharin 0.02% solution control started and was maintained for over 3 weeks. Mechanical allodynia was tested using von Frey filaments. (a) The mechanical sensitivity thresholds (PWT) of female mice is lower than that of male mice. However, both sexes developed mechanical allodynia similarly, and formoterol was effective in reversing the cuff-induced allodynia in both male and female mice.

  Figure 1(a)). The same antiallodynic effect was also present in MOP (Figure 1(c); F 3,23 = 23.82, p < 0.001) and KOP receptor-deficient mice (Figure 1(c); F 3,23 = 23.82, p < 0.001), while the terbutaline treatment was ineffective in DOP receptor-deficient mice (Figure 1(c); F 3,23 = 23.82, p < 0.001).

  (a); F 11,242 = 5.8, p < 0.001). The 0.001 µg/mL dose of formoterol had no significant effect; and treatments at different doses did not affect the contralateral nociceptive thresholds (Left Paw, Figure3(a)). The area the curve (AUC) for the treatment period is presented in Figure3(b). The drinking bottles were regularly weighed during the experiment. Considering the volume of solution drank by the mice per 24

  (a); Female mice: F 10,90 = 2.2, p < 0.01; Male mice: F 10,90 = 5.4, p < 0.001). Groups including the same number of male and female mice were pooled for data presentation (Figure 4(b), (c) and (d)).

  (b); F 10,210 = 5.6, p < 0.001). The same antiallodynic effect was also present in MOP (Figure 4(c); F 10,150 = 5.5, p < 0.001) and KOP receptor-deficient mice (Figure 4(c); F 10,150 = 4.1, p < 0.001), while formoterol treatment was ineffective in DOP receptor-deficient mice (Figure 4(c); F 10,150 = 2.7, p < 0.001), and in β 2 -AR deficient mice (Figure 4(d); F 10,150 = 1.8, p < 0.05).

Figure 1 .

 1 Figure 1. DOP receptors, but not MOP or KOP receptors, are critical to terbutaline treatment of

Figure 2 .

 2 Figure 2. Pharmacological blockade of DOP receptors induced a relapse of allodynia in

20 Figure 3 .

 203 Figure 3. Chronic oral formoterol treatment suppresses cuff-induced mechanical allodynia

Figure 4 .

 4 Figure 4. DOP receptors, but not MOP or KOP receptors, are critical to formoterol treatment of

  Males and females were then pooled in each experimental group, with an equal number of mice of both sexes in each group. (b, c) Chronic formoterol treatment suppressed the ipsilateral cuff-induced allodynia in wild-type mice, as well as in MOP and KOP receptor-deficient mice, but it remained ineffective in DOP receptor-deficient mice. (d) Formoterol had no action on mechanical allodynia in β 2 -AR deficient mice. (n = 3-4 males and 3-4 females per group, *p < 0.05 compared to Sham-operated control group drinking vehicle). Data are expressed as mean ± SEM.

21 Figure 5 .

 215 Figure 5. Pharmacological blockade of DOP receptors induced a relapse of allodynia in

  Figure 1 162x62mm (300 x 300 DPI)

  Mice were grouphoused two to five per cage and kept under a 12 hour light/dark cycle with food and water ad libitum. A total of 40 C57BL/6J mice, 44 MOP, 43 DOP, 44 KOP and 24 β 2 -AR-related mice were used for the experiments. Experiments were performed in agreement with European guidelines (EU 2010/63) and under protocols approved by the "Comité d'Ethique en Matière d'Expérimentation Animale de Strasbourg" (CREMEAS, CEEA35). At the end of the experiments, mice were killed by cervical dislocation according to institutional ethical guidelines. The animal facilities Chronobiotron UMS3415 are registered for animal experimentation under the Animal House Agreement A67-2018-38.

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Table 1 .

 1 Detailed Statistical analysis.

						Molecular Pain
	Supplemental Material
	Experiment Assessment	Treatment	Test	Groups (n)	Analysis	Statistics	Post-hoc	Figure
	Terbutaline treatment in WT mice	Mechanical PWT	NP surgery Chronic terbutaline	von Frey	Sham, Saline (8) Sham, Terbutaline (8) Cuff, Saline (9) Cuff, Terbutaline (9)	Group x Time Repeated measures ANOVA	F7,231=10.25, p<0.001	p<0.001: D4 to D35: "Cuff Saline" < "Sham Saline"; D28 to D35: "Cuff Saline" < "Cuff, Terbutaline"	1(a)
	Mechanical allodynia in WT, KOP -/-, MOP -/-, DOP -/-mice	Mechanical PWT	NP surgery Genotype	von Frey	Cuff, WT (9) Cuff, KOP -/-(6) Cuff, MOP -/-(7) Cuff, DOP -/-(6)	Group x Time Repeated measures ANOVA	F18,432=1.20, p=0.16	p<0.001: D0 > D2 to D36 for all groups	1(b)
					WT, Cuff Saline (9)
	Terbutaline treatment in opioid receptor deficient mice	AUC	NP surgery Chronic terbutaline		WT, Cuff Terbutaline (9) KOP -/-, Cuff Terbutaline (8) MOP -/-, Cuff Terbutaline (10)	Genotype x Treatment One-way ANOVA	F3,23=23.82, p<0.001	p<0.01: ("WT, Cuff Saline" = DOP -/-, Cuff Terbutaline) < ("WT, Cuff Terbutaline" = "KOP -/-, Cuff Terbutaline" = "MOP -/-, Cuff Terbutaline")	1(c)
					DOP -/-, Cuff Terbutaline
					(12)
	Effect of naltrindole on terbutaline treatment in WT mice	Mechanical PWT	NP surgery Chronic terbutaline + acute naltrindole	von Frey	Sham, Saline (8) Sham, Terbutaline (8) Cuff, Saline (9) Cuff, Terbutaline (9)	Group x Time Repeated measures ANOVA	F1,33=18.83, p<0.001	p<0.01: T30 min: ("Cuff Terbutaline" = "Cuff Saline") < ("Sham Saline" = "Sham Terbutaline")	2(a)
	Effect of naltrindole on terbutaline treatment in MOP -/-mice	Mechanical PWT	NP surgery Chronic terbutaline + acute naltrindole	von Frey	Sham, Saline (9) Sham, Terbutaline (8) Cuff, Saline (12) Cuff, Terbutaline (10)	Group x Time Repeated measures ANOVA	F1,21=21.61, p<0.001	p<0.05: T30 min: ("Cuff Terbutaline" = "Cuff Saline") < ("Sham Saline" = "Sham Terbutaline")	2(a)
	Effect of naltrindole on terbutaline treatment in KOP -/-mice	Mechanical PWT	NP surgery Chronic terbutaline + acute naltrindole	von Frey	Sham, Saline (9) Sham, Terbutaline (8) Cuff, Saline (9) Cuff, Terbutaline (8)	Group x Time Repeated measures ANOVA	F1,20=5.05, p<0.01	p<0.01: T30 min: ("Cuff Terbutaline" = "Cuff Saline") < ("Sham Saline" = "Sham Terbutaline")	2(a)
	Peripheral opioid receptors and	Mechanical PWT	NP surgery Chronic terbutaline +	von Frey	Sham, Saline (8) Cuff, Terbutaline (9)	Group x Time Repeated measures ANOVA	F1,20=10.90, p<0.01	p<0.01: T30 min: "Cuff Terbutaline" < "Sham Saline"	2(b)

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Funding

This work was supported by the Centre National de la Recherche Scientifique [contracts UPR3212 and UMR5293], the University of Strasbourg and the Agence Nationale de la Recherche [Euridol ANR-17-EURE-0022]; MK was supported by a CNRS disability doctoral fellowship, the Fondation d'Entreprise Banque Populaire and the Fédéeh Hewlett Packard. We are grateful to Profs. Brigitte Kieffer and Claire Gavériaux-Ruff (Strasbourg, France) for providing MOP, DOP and KOP related mouse breeders. Page 15 of 29 https://mc.manuscriptcentral.com/molecularpain Molecular Pain

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The authors declare that they have no competing interests.