MSc Meggane Melchior

PhD Pierre-Eric Juif

MSc Géraldine Gazzo

Nathalie Petit-Demoulière

Virginie Chavant

Adrien Lacaud

Yannick Goumon

Alexandre Charlet

Vincent Lelièvre

PhD Pierrick Poisbeau
email: poisbeau@inci-cnrs.unistra.fr

MSc Nathalie Petit-Demouliere

MSc Virgine Chavant

Yannick Tech

PhD Alexandre Goumon

PhD Vincent Charlet

PhD Pierrick Lelièvre

Prof. Pierrick Poisbeau

Juif Pe

Petit-Demoulière N

Chavant

Goumon

Charlet

& Poisbeau

Pharmacological rescue of nociceptive hypersensitivity and oxytocin analgesia impairment in a rat model of neonatal maternal separation

Keywords: early life stress, spinal nociceptive porcessing, hyperalgesia, oxytocin, allopregnanolone, HDAC inhibitor, epigenetic

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

RESUME

Oxytocin (OT), known for its neurohormonal effects around birth, has recently been suggested for being a critical determinant in neurodevelopmental disorders. This hypothalamic neuropeptide exerts a potent analgesic effect through an action on the nociceptive system. This endogenous control of pain has an important adaptive value but might be altered by early life stress (ELS), possibly contributing to its long-term consequences on pain responses and associated comorbidities. We tested this hypothesis using a rat model of neonatal maternal separation (NMS) known to induce long term consequences on several brain functions including chronic stress, anxiety, altered social behavior and visceral hypersensitivity. We found that adult rats with a history of NMS were hypersensitive to noxious mechanical/thermal hot stimuli and to inflammatory pain. We failed to observe OT receptor-mediated stress-induced analgesia as well as OT anti-hyperalgesia after carrageenan inflammation. These alterations were partially rescued if NMS pups were treated by intraperitoneal daily injection during NMS with OT or its downstream second messenger allopregnanolone. Involvement of epigenetic changes in these alterations was confirmed since neonatal treatment with the histone deacetylase (HDAC) inhibitor SAHA, not only normalized nociceptive sensitivities, but also restored OT receptor-mediated stress-induced analgesia as well as the endogenous anti-hyperalgesia in inflamed NMS rats. There is growing evidence in the literature that ELS might impair the nociceptive system ontogeny and function.

This study suggests that these alterations might be restored while stimulating OT receptor signaling or HDAC inhibitors, using molecules that are currently available or part of clinical trials for other pathologies.

INTRODUCTION

Oxytocin (OT) is a nonapeptide synthesized by hypothalamic neurons mainly located in the paraventricular (PVN) and supraoptic (SON) nuclei [START_REF] Poisbeau | Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels[END_REF]. OT acts both as a neurohormone when released into the blood via the posterior pituitary or as a neurotransmitter when synaptically released into the spinal cord (SC) and supraspinal regions. Recently, our laboratory identified a small population of 30 parvocellular neurons in the PVN which coordinate the peripheral and spinal release of OT to limit inflammatory pain symptoms [START_REF] Eliava | A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing[END_REF]. Indeed parvocellular OT neurons of the PVN trigger OT secretion in superficial and autonomic layers of the SC where OT receptors are found [START_REF] Poisbeau | Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels[END_REF]. OT displays anti-nociceptive properties by increasing GABAergic inhibition in the SC thus decreasing excitability in superficial and deep SC neurons [START_REF] Poisbeau | Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels[END_REF]. In addition to these central-related effects, a peripheral component of OT-mediated analgesia via a direct effect at the dorsal root ganglion (DRG) level has been documented [START_REF] Moreno-Lopez | Identification of oxytocin receptor in the dorsal horn and nociceptive dorsal root ganglion neurons[END_REF].

Activation of the OT analgesic system occurs under different conditions such as inflammatory sensitization [START_REF] Eliava | A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing[END_REF], neuropathic pain [START_REF] Martinez-Lorenzana | PVN electrical stimulation prolongs withdrawal latencies and releases oxytocin in cerebrospinal fluid, plasma, and spinal cord tissue in intact and neuropathic rats[END_REF], or forced swim stress (FSS) [START_REF] Wotjak | Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons[END_REF]. In the carrageenan model of inflammatory sensitization, spinal OT levels are increased for at least 24 h and OT receptormediated local production of the neurosteroid allopregnanolone (AP) leads to a sustained GABAergic activity to maintain spinal antinociceptive control [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF]. After a non-noxious FSS, OT is also responsible for significant and transient stress-induced analgesia (SIA) [START_REF] Wotjak | Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons[END_REF]. In this context, a deficit in OT signaling might be related to neurodevelopmental brain pathologies such as autism spectrum disorders [7; 8].

The development of the nociceptive system starts before birth and follows a similar pattern in the rat and in the human fetus [9; 10]. It is well accepted that the developmental stage of the rat just after birth is similar to that of the human fetus at the beginning of the 3 rd trimester of gestation.

Descending controls of pain mature during the first 3 weeks after birth in the rat [START_REF] Fitzgerald | The development of nociceptive circuits[END_REF] and begin to mature during the end of the 3 rd semester in humans [START_REF] Hartley | Changing Balance of Spinal Cord Excitability and Nociceptive Brain Activity in Early Human Development[END_REF]. At the SC level, spinal inputs and chloride homeostasis undergo re-organization steps after birth to be fully matured [13; 14] making them possibly vulnerable to early adverse events. Neonatal maternal separation (NMS) is a wellestablished model of early life stress (ELS) that induces long term neuroendocrine and behavioral changes in rodents, with impact on the HPA axis, social and cognitive behaviors, and sensory and nociceptive systems [15; 16]. Long-term consequences of NMS are indeed visceral hypersensitivity [START_REF] Sengupta | Visceral pain: the neurophysiological mechanism[END_REF], epigenetic changes, and associated sensory dysfunctions [18; 19]. Recently, a chronic adult treatment with histone deacetylase (HDAC) inhibitor was shown to be efficient to counteract NMS-induced visceral hypersensitivity [START_REF] Moloney | Early-life stress-induced visceral hypersensitivity and anxiety behavior is reversed by histone deacetylase inhibition[END_REF].

In this study, we used behavioral measures and in vivo electrophysiological recordings from SC dorsal horn neurons to characterize the long-term consequences of NMS on the oxytocinergic descending control of pain. Then, we aimed to prevent the development of NMS pain phenotype using pharmacological tools, targeting the OT system or epigenetic factors early during the postnatal period.

MATERIALS AND METHODS

Animal care and neonatal maternal deprivation procedure

Female Wistar rats with litters were housed in a temperature-controlled room (22°C) under a 12h light-dark cycle (lights on at 07:00 AM), with ad libitum access to food and water. All experiments were conducted in conformity with the recommendations of the European Committee Council Direction of September 22, 2010 (2010/63/EU), with an authorization for animal experimentation from the French Department of Agriculture (License 67-116 to PP) and with a procedure agreement evaluated by the regional ethical committee (CREMEAS authorization number APAFIS#7678-20 16112223027528 v2).

At birth, litters were randomly assigned to two groups: Neonatal NMS and non-separated control litters. The litters allocated to the NMS group were removed from the nest cages 3 h per day from postnatal days 2 (P2) to 12 (P12) and placed on a heating pad (37°C) in a separate cage. The litters allocated to the control group remained with their mother in their home cages during the entire NMS interval and received no special handling other than that necessary to change the bedding of their cage. Pups were weaned at P21 and housed four rats per cage before being randomly chosen at 8 weeks or more for behavioral analysis or electrophysiological recordings. In each experiment, animals were chosen randomly in at least two different litters. Both males and females were used in this study, as no sex specific differences could be seen for the different neurophysiological and behavioral measures.

Measure of basal mechanical and thermal nociception

Mechanical nociception was measured on adult rats with a calibrated forceps (Bioseb, Vitrolles, France) as previously described [START_REF] Oe | Calibrated forceps: a sensitive and reliable tool for pain and analgesia studies[END_REF]. Briefly, the habituated rat was loosely restrained with a towel masking the eyes in order to limit stress by environmental stimulations. The tips of the forceps were placed at each side of the paw and a gradually increasing force was applied. The pressure producing withdrawal of the paw corresponded to the nociceptive threshold value. Thermal nociception was measured by a blind experimenter with the Hargreaves method, as previously described [START_REF] Hargreaves | A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia[END_REF]. Briefly, rats were placed in plexiglas boxes (20cm x 25cm x 45cm) for a 15-20 minutes habituation period and then hindpaws were exposed to a radiant heat and the latency time required to induce paw withdrawal was recorded and considered as the nociceptive heat threshold. For both tests, measurements were performed three times for each hindpaw and values were averaged.

Forced swim stress

At adulthood, rats were exposed to a forced swim stress procedure (FSS) in a Plexiglas® cylinder (diameter: 30 cm; wall height: 60 cm), filled with water (height: 40 cm) at a temperature of 20°C for 10 minutes. After FSS exposure, animals were gently dried. Mechanical thresholds were measured before (baseline) and 15 min after FSS in both NMS and control animals. This protocol has been described to be associated with a plasmatic and spinal release of oxytocin [START_REF] Wotjak | Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons[END_REF]. The oxytocinergic contribution of FSS to SIA was confirmed in a subset of rats receiving an intrathecal (i.t) injection of 10µL of OTR antagonist dOVT ((d(CH₂)₅¹,Tyr(Me)²,Thr⁴,Orn⁸,des-Gly-NH₂⁹)-Vasotocin, Bachem, Germany) at 200µM or 10µL saline 30 minutes before FSS.

Carrageenan Inflammatory pain model

To induce a painful inflammatory sensitization in the rat, 150µL of carrageenan (3% in NaCl 0.9%, Sigma Aldrich, Germany) were injected in the plantar surface of the right hindpaw for behavioral experiments, whereas injections in both hindpaws were performed for electrophysiological.

Control animals received either similar injections of NaCl 0.9% or no injection.

In vivo recording of spinal cord neurons and analysis Male and female rats were anaesthetized with isoflurane (2 %; pushed by pressurized air at a flow rate of 0.5 L.min -1 ) and body temperature was regulated using a thermostatically controlled heating blanket (Harvard Apparatus Ltd, France) maintaining the body temperature at 37°C. Animals were set up in a stereotaxic frame (La Précision Cinématographique, Eaubonne, France), with the cervical and sacral vertebrae firmly held. The lumbar SC (L4-L5) was exposed by laminectomy. After removal of the meninges, the SC surface was covered with a thin isolating layer of mineral oil. This opening allowed us to record SC neurons and apply several drugs directly on the SC. At the end of the experiment, animals were killed with an overdose of isoflurane.

Single unit extracellular in vivo recordings were made from wide dynamic range (WDR) neurons, located in lamina V and receiving peripheral sensory messages mediated by Aβ, Aδ and C-type primary afferent fibers. In the lumbar enlargement of the SC, we monitored voltage changes through stainless microelectrodes (FHC, Bowdoin, USA) connected to an extracellular differential amplifier (DAM-80, WPI, Aston, UK). Electrical signals were then digitized (Power 1401 CED, Cambridge, UK), processed with the Spike 2 software (CED Cambridge, UK). Neurons were characterized by their responses to transcutaneous electrical stimulation of the hindpaw receptive field as previously described [START_REF] Juif | Neurohormonal effects of oxytocin and vasopressin receptor agonists on spinal pain processing in male rats[END_REF]. Electrical stimulation was applied through two thin cutaneous pin electrodes placed in the center of the receptive field on the hindpaw. Increasing intensities were applied from 1 V to C fiber activation threshold (duration of 1 ms). Trials consisted of a train of 90 electrical stimuli (3 x C-type fiber threshold) applied repetitively (1 Hz) to the excitatory receptive field. This method has been usually used to obtain a stable and reproducible "wind up" [START_REF] Mendell | Physiological properties of unmyelinated fiber projection to the spinal cord[END_REF], a property of C-type nociceptive fibers. The electrically-evoked response of the neuron was captured and displayed as a post-stimulus histogram, for A-type and C-type discharge. Action potentials appearing early after the stimulus artifact were classified as A-type (0-50 ms) whereas the delayed response corresponded to C-type (50-800 ms).

Spinal cord sampling and PCR analysis

Lumbar spinal cord samples were collected, reconstituted in a guanidine thiocyanate/βmercaptoethanol preparation using ultraturax and stored at -80°C. Total RNA was extracted according to a protocol derived from the original procedure of Chomczynski and Sacchi [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF], consisting of two independent total RNA extractions separated by a DNAseI treatment ((TURBO DNaseTM, Ambion, Life technologies, Saint Aubin, France), as previously described in detail [START_REF] Lelievre | Fibroblast growth factor-2 converts PACAP growth action on embryonic hindbrain precursors from stimulation to inhibition[END_REF]. 800ng RNA were used for reverse transcription with the RT iScript kit (Bio-Rad, Marnes-la-Coquette, France). Quantitative PCR was performed using SYBR Green Supermix (Bio-Rad), on the iQ5 Real Time PCR System (Bio-Rad). Amplifications were carried out in 42 cycles (20s at 95°C, 20s at 60°C and 20s at 72°C). Standardization was made possible using standard curves made from serial dilutions of samples, and amplification data is shown as relative gene expression, calculated as the ratio between cDNA concentration of the gene of interest and that of the housekeeping gene. Preliminary experiments showed that among the different classic housekeeping genes routinely analyzed in the laboratory, and compared with 18S ribosomal RNA, hypoxanthin-guanine phosphoribosyltransferase (HPRT) transcripts remained highly stable among the different samples.

Therefore, HPRT was selected as the housekeeping gene to analyze and evaluate potential changes in gene expression in all experiments presented here. To specifically amplify mRNA encoding various rat proteins, we designed specific primer sets (sense and antisense respectively) using Oligo6.0 and M-fold softwares. Primer sequences for all genes of interest can be found in supplementary table 1. Samples were accurately dispensed in duplicates using a robotic workstation (Freedom EVO100, Tecan, Lyon, France), and amplification efficacy given by standard curves was always close to 100% (± 2%), whilst amplification specificity was assessed by a melting curve study.

Neonatal rescue procedure

To reverse NMS long term consequences on pain behavior, a pharmacological neonatal treatment was performed in NMS pups. During NMS protocol, all pups in the litter were injected with the same molecule and then tested at adulthood to investigate the long-term efficiency of neonatal treatment. OT (Bachem, Weil am Rhein, Germany), the HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid, Vorinostat, ApexBio Technology, Houston, USA), or vehicle (NaCl, oil or 10% DMSO respectively) were injected intraperitoneally (i.p. 10µL) at 1mg/kg and 5mg/kg respectively once daily (o.d) during the NMS procedure (i.e from P2 to P12). In another subset of animals, allopregnanolone (AP, Santa Cruz biotechnology, Dallas, USA) was injected subcutaneously at 10mg/kg once every two days during the NMS procedure. To avoid unnecessary supplementary handling of the pups, injections were performed at the beginning of the separation process. Pups were then normally weaned at P21, and no special handling was performed until P45, when the efficiency of neonatal treatment was assessed for each neonatal group using behavioral testing.

First, mechanical thresholds were measured prior (baseline) and after FSS (SIA measurement).

Then one week later, the efficacy of the OT descending control of pain after carrageenan inflammation of the hindpaw was measured with the same protocol as the one previously described (using spinal dOVT injection 24H after inflammation).

Drugs and Treatments

OT receptor (OTR) agonists and antagonist, as well as V1aR antagonist were prepared as a concentrated solution in water (1mM) and diluted to the needed concentration in NaCl (0.9%) before experiment. The selective OTR antagonist dOVT (d(CH2)5-Tyr(Me)-[Orn8]-vasotocine) was used at a final concentration of 1 µM in Saline (NaCl 0.9%). dOVT was injected i.t. (volume 20 µL) or directly on the spinal cord in electrophysiological recordings (volume 500 µL). V1aR antagonist [Phenylacetyl1,O-Me-D-Tyr2,Arg6,8,Lys9]-vasopressinamide (AV1AR: Sigma Aldrich, Saint-Louis, France) was injected i.t at a concentration of 1µM (volume 20µL). OT and TGOT ((Thr⁴,Gly⁷)-Oxytocin) (Bachem, Weil am Rhein, Germany) were used as an agonists for OTRs. OT was dissolved in saline and injected i.t or i.p. at a concentration of 10 µM or 1mg/kg, respectively. TGOT was also dissolved in NaCl 0.9% and injected at a concentration of 1 µM (i.t.). Allopregnanolone and SAHA were freshly prepared before the experiment. Allopregnanolone was used at 10mg/kg and dissolved in mineral oil by sonication at 4°C. The non-selective HDAC inhibitor SAHA was dissolved in 10% DMSO and injected i.p. at a concentration of 5mg/kg.

Statistical analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 6 software (LaJolla, CA, USA). For each experiment, parametric analysis was used only after verifying the normal distribution of the values. Unpaired Student ttest was used to compare baseline nociceptive sensitivity and OTR and oxytocinase (OTase) expression in NMS and CTRL rats. Repeated measures Two-Way ANOVA followed by Sidak post hoc test was used to analyze inflammatory symptoms and to analyze wind-up efficiency. Repeated measures Two-Way ANOVA followed by Tukey's post hoc test was used to evaluate the effect of OT and TGOT spinal application in control and NMS rats. One way ANOVA and Tukey post-hoc tests were used to compare the effects of spinal application of dOVT in control or NMS rats, to evaluate the efficiency of SIA in both groups, to analyze the success of the rescue experiments on baseline nociceptive sensitivities and to evaluate spinal mRNA expression of 3α-hydroxysteroid dehydrogenase (3α-HSD), 5α-reductase type II (5α-red II), P450scc and translocator protein (TSPO). Mann-Whitney test was used to compare the effects of spinal application of the V1aR antagonist in control and NMS rats. Kruskal Wallis (KW) followed by Dunn's post hoc test was used to compare wind-up frequencies and spinal expression of histone deacetylases (HDAC) transcripts.

Differences were considered to be statistically significant for p < 0.05.

RESULTS

Consequences of NMS on the nociceptive sensitivity and on the efficacy of oxytocinergic SIA

We first characterized the long-term effects of NMS on baseline mechanical and thermal nociceptive thresholds (Figure 1A), as well as the intensity of stress-induced mechanical analgesia (SIA) measured after FSS (Figure 1B). Compared to the control group, NMS animals exhibited hypersensitivity to both mechanical and thermal stimulation at adulthood (P>100). NMS rats displayed significantly lower mean pressure thresholds (control: 445.5 ± 6.1 g, N = 12; NMS: 268.5

Figure 1 near here

Previous studies showed that both central and peripheral release of OT contributed to SIA after a FSS [26; 22]. Because NMS is known to alter the normal reactivity to stress and lead to a dysfunction of the HPA axis, we investigated the efficacy of the oxytocinergic component of SIA in NMS animals (Figure 1B). As expected, the FSS paradigm induced a significant analgesia 20 minutes after swim in the control group (Figure 1B). Mean baseline-corrected mechanical thresholds were increased by 239 ± 12.3 g in the control group (N=8) but only by 10.7 ± 23.4 g in the NMS group (N=8). An injection of dOVT prior to FSS decreased SIA intensity to 56.9 ± 20.1 g in the control group (N=8) and had no effect in the NMS group (N=8) where it varied of 20.75 ± 20.7 g (p < 0.001, One-Way ANOVA, F = 28.9). These results suggest a blunted adaptation to stressful conditions in NMS rats associated with possible dysfunction of the oxytocinergic descending control of pain.

Behavioral response to a painful inflammatory sensitization by carrageenan

We next compared the intensity and time course of inflammatory pain symptoms triggered by an intraplantar hindpaw injection of carrageenan to possibly highlight differences in the expression of mechanical/thermal hyperalgesia in control and NMS rats (Figure 2). In both groups, carrageenan injection induced a sustained mechanical and thermal hyperalgesia which peaked 7 h after injection, as previously described [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF]. Although basal mechanical nociceptive thresholds (i.e., before carrageenan injection) were significantly lower in NMS rats, the time course of mechanical hyperalgesia was not significantly different between both groups (Figure 2A1-B1). In the control group, mean pressure threshold dropped from 445.8 ± 18.7 g to 30.1 ± 5.1 g, 7 h after injection (N = 6; repeated measures two-way ANOVA, treatment x time, F(12,120) = 63.81, p < 0.001) and mechanical hyperalgesia lasted for at least 6 days for control. As mentioned above, at baseline, NMS rats exhibited a lower mean pressure threshold of 295.5 ± 4.8 g (N = 6), which further decreased to 70.8 ± 12.6 g, 7 h after carrageenan injection. As illustrated in Figure 2B1, hyperalgesia was detected at a significant level for 6 days (N = 6; repeated measures two-way ANOVA, treatment x time, F(12,120) = 30.78, p < 0.001). Concerning thermal hot sensitivity, 7 h after carrageenan injection mean latencies decreased to 3 .3 ± 0.6 s and 4.2 ± 0.8 s in control and NMS rats (N=6 per group) respectively. Interestingly, thermal hot hyperalgesia persisted for at least 1 days for control rats and up to 4 days in NMS rats (Figure 2B2) (repeated measures two-way ANOVA, treatment x time, F(8,80) = 13.67, p < 0.001 for CTRL and F(10,100) = 10.69, p < 0.001 for NMS).

Figure 2 near here

Oxytocinergic contribution to the intensity of carrageenan-induced pain symptoms and to the spinal cord processing of nociceptive messages

In the carrageenan model of inflammatory sensitization, spinal concentrations of OT are elevated during the early phase of inflammation and have been shown to limit pain sensitivity [START_REF] Juif | Neurohormonal effects of oxytocin and vasopressin receptor agonists on spinal pain processing in male rats[END_REF]. To investigate whether NMS affects OTergic anti-hyperalgesia, i.t application of the selective OTR antagonist dOVT was performed 24 hours after carrageenan injection. As OT may act via the activation of vasopressin V1a receptors (V1ARs) at high concentrations, we also examined a possible contribution of these receptors using i.t application of V1aR selective antagonist.

In the control group and as previously shown [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF], a single i.t injection of dOVT (1µM, 20µL), induced a rebound of mechanical and thermal hyperalgesia. Anti-hyperalgesia mediated by OTR could be seen in the CTRL group as a decrease in the mean baseline-corrected mechanical threshold 4 h (i.e. 28h after carrageenan) after dOVT injection by -75.6 ± 8.4 g (N= 10 Figure 3A). This decrease was absent in the saline-treated group (-2 ± 8.4 g, N=10) (One-way ANOVA F = 17.5, p < 0.001). For thermal withdrawal latency (Figure 3B), OTR anti-hyperalgesia could also be seen as a significant decrease in the mean values after dOVT compared to saline (N = 10; One-way ANOVA, F = 9.3, p < 0.001). This corresponded to a change of -3.9 ± 0.6 s (N=10) and of 1.1 ± 0.8 s (N=10), for the dOVT and saline groups, respectively. Intrathecal injection of the V1aR antagonist was without effect on mechanical (before: 155.25 ± 3.2 g; after i.t. injection: 177.25 ± 18.2 g, N = 4; Mann-Whitney test: U = 4.0; p = 0.3429) or thermal nociceptive threshold (before: 10.0 ± 0.9 s; after i.t. injection: 11.0 ± 0.9 s, N=4; Mann-Whitney test: U = 5.0; p = 0.4571) in the control group of rats.

Both results confirmed OTR activation to be responsible for a limitation of pain symptoms early after carrageenan injection in control animals. Interestingly, there were no changes in the mean mechanical (dOVT: delta of 5.8 ± 10.8 g, N=8; saline: delta of -27.2 ± 9 g, N=7; One-way ANOVA F=17.5 p<0.001) and thermal hyperalgesia values (dOVT: delta of -1 ± 0.7 s, N=8; saline: delta of 0.1 ± 0.7 s, N=7; One-way ANOVA F=9.3 p<0.001) in NMS animals. As for control rats, a single i.t. injection of the V1aR antagonist did not change mechanical (before: 164.65 ± 9.9 g; after i.t. Together, this suggests that NMS has long-term consequences on the efficacy of OTR-mediated spinal analgesia, at least in short-term inflammatory painful sensitization.

Figure 3 near here

To further characterize this NMS-altered OTR-mediated analgesia in NMS adult rats, we recorded WDR neurons in the SC of anesthetized rats and analyzed the spinal processing of nociceptive messages through the expression of windup, a nociceptive-specific short-term plasticity [START_REF] Herrero | Wind-up of spinal cord neurones and pain sensation: much ado about something?[END_REF]. As illustrated in figure 4, windup recorded 24 h after carrageenan injection was significantly amplified after the spinal application of dOVT (1µM). In control animals (N=6, figure 4A), windup protocol led to an average maximum frequency of 23.6 ± 1.5 spikes/s. This value significantly increased to a maximum frequency of 44.1 ± 4.8 spikes/s after dOVT application (N = 6; two-way repeated measures ANOVA, treatment x time, F(14,70) = 7.811; p < 0.0001). As shown in figure 4B, we did not see any change in windup frequencies in NMS animals (N=6) after dOVT application (before application: 26.8 ± 4.3 spikes/s; after application: 25 ± 4.1 spikes/s; two-way repeated measures ANOVA, treatment x time, F(14,70) = 1.366; p = 0.1936). Summary of results can be found in figure 4C (Kruskall-Wallis test: KW =8.9; p = 0.03).

Figure 4 near here

Altogether, electrophysiological and behavioral experiments suggested that the OT descending inhibitory control of pain is inefficient in NMS rats.

OTR expression and OTR-induced analgesia

In order to better understand the possible molecular and cellular alterations induced by NMS, we first analyzed the expression of OTR and a possible dysfunction of its downstream signaling, focusing on the SC as it is the first relay for nociceptive messages. There were no difference in the spinal expression of OTR gene transcripts between control (N=7; 3.1 ± 0.9 spinal relative expression) and NMS (N=8; 2.4 ± 0.2 spinal relative expression) rats (unpaired Student's t-test, p = 0.44, t = 0.78, df = 13), as illustrated in figure 5A.

Figure 5 near here

To further confirm that OTR were not only expressed at similar levels in both groups but were equally functional, we measured the analgesic behavioral response to i.t injection of OT (1µM 20µL) or to the selective agonist TGOT (1µM 20µL) in CTRL and NMS rats (Figure 5B). In CTRL animals OT (N = 8) or TGOT (N = 8) significantly increased mechanical thresholds 20 minutes after the injection (OT: from 467.4 ± 19.4 g to 741.6 ± 67.1 g; TGOT: from 471.8 ± 14.6 g to 813.2 ± 26.3 g; Two-way repeated measures ANOVA, treatment x time, F(4,40) = 14.67; p < 0.0001). Both OT (N = 7) or TGOT (N = 8) significantly increased mechanical thresholds 20 minutes after the injection in a reversible manner in NMS animals (OT: from 288.4 ± 5.0 g to 533.9 ± 13.1 g; TGOT: from 246.7 ± 11.2 g to 460.1 ± 17.3 g; Two-way repeated measures ANOVA, treatment x time, F(11,38) = 66.94; p < 0.001). This result confirmed that spinal OTR are functional in NMS rats and capable of producing efficient analgesia. We thus checked whether NMS could lead to an increase in the spinal degradation of OT. As illustrated in Supplementary Figure 1A, we found no difference in the spinal expression of oxytocinase (OTase) mRNA between control and NMS rats (N=8; unpaired Student t-test, t = 0.1317, df = 14; p = 0.8971).

Neonatal treatments with OT and allopregnanolone

Many studies provided evidence that OT in the neonatal period could be protective against early life adverse events or lesions [28; 29]. Moreover, NMS is known to reduce maternal behavior towards the pups and a subsequent altered development of OT systems, possibly leading to blunted OT levels [START_REF] Lovic | Maternally separated rats show deficits in maternal care in adulthood[END_REF]. This includes molecular mechanisms affecting chloride homeostasis and the production of neuroprotective neurosteroids such as allopregnanolone [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF].

In a first set of experiments, we treated NMS rat pups o.d. from P2 to P12 with OT (1mg/kg in 0.9% NaCl, i.p., Figure 6). As shown in figure 6A, neonatal OT treatment fully restored normal mechanical nociceptive thresholds when measured at P50 (control: 320.7 ± 10.9 g, N = 12; NMS: 235.1 ± 6.9 g, N = 8; OT-treated NMS: 320.4 ± 6.3 g, N = 10; One-way ANOVA, F = 23.39, p < 0.001). OT neonatal treatment also restored an efficient analgesia after FSS (Figure 6B; delta for Control group: 261.8 ± 18.6 g, N = 9; NMS: 17.8 ± 11.6 g, N = 8; OT-treated NMS: 168.9 ± 10.4 g, N = 9; One-way ANOVA, F = 30.08, p < 0.001). In contrast to SIA after FSS, neonatal OT treatment failed to restore OT anti-hyperalgesia 24 hours after carrageenan-induced inflammation, measured as before using i.t dOVT injection (Figure 6C). The dOVT injection did not significantly amplified mechanical hyperalgesia (delta for NMS: 5.8 ± 10.9 g, N = 8; OT-pretreated NMS: 45.6 ± 19.7 g, N = 8; One-Way ANOVA, F = 14.28, p < 0.001).

Figure 6 near here

Recently, an original long-lasting molecular mode of action of OT in SC neurons has been proposed. It leads to the stimulation of AP synthesis, a neurosteroid well-known for its analgesic and neuroprotective roles [START_REF] Poisbeau | Analgesic strategies aimed at stimulating the endogenous production of allopregnanolone[END_REF]. We thus tried to restore OT function in NMS rats by submitting newborn pups to AP during the NMS period. As illustrated in the different panels of figure 6, a neonatal treatment with s.c AP (10mg/kg) between P2 and P12 was associated with a mean mechanical threshold at adulthood which was higher (i.e., hyposensitivity) than those measured in control animals (Figure 6A; Control: 320.7 ± 10.9 g, N = 12; AP-treated NMS: 394.1 ± 21.9 g, N = 10; One way ANOVA, F = 23.39, p < 0.001). As for neonatal OT treatment, SIA could be restored by AP treatment (Figure 6B; baseline corrected mean mechanical threshold in AP-treated NMS: 351 ± 47 g, N = 8, One Way ANOVA, F = 30.08, p<0.001). As previously reported, endogenous OT anti-hyperalgesia could not be revealed after dOVT i.t. injection (Figure 6C; APtreated NMS: -23 ± 16 g, N = 8; One Way ANOVA, F = 14.28, p < 0.001). Taken together, our results likely suggested NMS to be associated with a deficit in OT signaling. However, we found no significant difference in the spinal transcripts levels of 3α-hydroxysteroid dehydrogenase (3α-HSD), 5α-reductase type II (5α-red II), nor cholesterol side-chain cleavage enzyme (P450scc), enzymes involved in AP synthesis (Supplementary Figure 1B; N = 8; One way ANOVA, F = 0.7409, p = 0.6384). Similarly, spinal translocator protein (TSPO) mRNA levels remained unchanged in NMS rats compared to controls.

NMS-induced epigenetic alterations and rescue of MS pain phenotype by targeting histone deacetylases

The long-term consequences of NMS on pain sensitivity and on the rat ability to cope with stress/pain potentially rely on epigenetic mechanisms. Indeed, several reports highlight the effects of NMS on the HPA axis and on maternal behavior [32; 33]. We thus measured the spinal expression of a set of transcripts coding for several epigenetic factors at different time points (from P7 to P100) in NMS and control animals (Figure 7A). We found that mRNA levels for some class I (HDAC 1 and 8) and class II HDAC (HDAC 7) were significantly increased in NMS animals at P24 (HDAC 1 and 7), P45 (HDAC 1, 7 and 8) and until P100 for HDAC 7 (Kruskal-Wallis; HDAC1 : KW = 60.33 , p < 0.0001 , N = 7 ; HDAC 7 : KW = 61.41 , p < 0.0001 , N = 7 ; HDAC 8 : KW = 49.28 , p < 0.0001 , N = 6).

In particular, HDAC 7 mRNA spinal expression was significantly increased in NMS animals compared to controls at P24 (0.49 ± 0.04 relative expression in CTRL and 1.22 ± 0.11 in NMS), as illustrated in figure 7B, and levels remained significantly higher until adulthood at P100 (0.36 ± 0.03 relative expression in CTRL and 1.01 ± 0.04 in NMS). These results likely suggest that NMS triggers long lasting changes in gene expression, involving at least HDAC functions.

Figure 7 near here

We hence performed a neonatal chronic treatment from P2 to P12 with SAHA (10 mg/kg i.p.), a non-selective HDAC I and II inhibitor. Neonatal treatment of newborn pups with SAHA during NMS was associated with mean mechanical nociceptive thresholds at adulthood (P50) which were similar to the control group (Figure 7C; Control: 320.7 ± 10.9 g, N = 8; SAHA-treated NMS: 303.8 ± 9.4 g, N = 10; One way ANOVA, F = 14.28, p < 0.001). This treatment also partially restored SIA after FSS (Figure 7D; SAHA-treated: 130 ± 26.7 g, n = 7; One Way ANOVA, F = 34.73, p < 0.001).

Interestingly, SAHA treatment partially restored OT anti-hyperalgesia after carrageenan inflammation (Figure 7E; Control: 75.6 ± 8.4 g; SAHA-treated: -41.7 ± 12.3 g; One-Way ANOVA, F = 15.75, p < 0.0001).

DISCUSSION

In summary, we provide functional evidence indicating that NMS strongly affects adult nociception and the efficacy of the oxytocinergic descending inhibitory control. NMS is associated with (i) hypersensitivity to mechanical and thermal hot stimulation at adulthood, (ii) increased vulnerability to thermal inflammatory-induced hyperalgesia, and (iii) dysfunction of the OT descending control of pain after non-painful FSS or painful inflammatory sensitization. We further succeeded in rescuing these abnormalities in the nociceptive phenotype by repeated neonatal injections of OT, AP, or SAHA. Altogether, our data suggest that NMS has a strong impact on nociceptive integration, which can blunt the efficacy of descending controls of pain such as the oxytocinergic system which targets the spinal GABAergic inhibitory system. It also suggests that the OT or epigenetic systems can be good targets to develop early treatments to counteract the negative effects of ELS, at least in this model.

Early life events promote nociceptive hypersensitivity and alter OTR-mediated analgesia

Early life stress includes a wide range of adverse events which have long-term negative effects on brain function. In humans, ELS has been associated with an increased risk of developing chronic pain states at adulthood [START_REF] Davis | Are reports of childhood abuse related to the experience of chronic pain in adulthood? A meta-analytic review of the literature[END_REF], including irritable bowel syndrome [35; 36]. In animal studies, the visceral hypersensitivity following NMS has been widely demonstrated, and correlated with differential activation of supraspinal regions, including the periaqueductal gray, which is a key area for descending pain controls. Here, we confirmed that nociceptive hypersensitivity extends to somatic mechanical and thermal hot stimuli, as previously published by our laboratory [START_REF] Juif | Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation[END_REF] and in other ELS models [START_REF] Takatsuru | Maternal separation decreases the stability of mushroom spines in adult mice somatosensory cortex[END_REF][START_REF] Pierce | Vaginal hypersensitivity and hypothalamicpituitary-adrenal axis dysfunction as a result of neonatal maternal separation in female mice[END_REF][START_REF] Yasuda | Maternal Separation Induces Orofacial Mechanical Allodynia in Adulthood[END_REF][START_REF] Amini-Khoei | Experiencing neonatal maternal separation increased pain sensitivity in adult male mice: Involvement of oxytocinergic system[END_REF]. Our observation of an increased thermal pain symptom after carrageenan is consistent with studies showing an increased response after formalin injection [START_REF] Uhelski | Maternal separation stress leads to enhanced emotional responses to noxious stimuli in adult rats[END_REF] or after partial sciatic ligation [START_REF] Nishinaka | Sex differences in depression-like behavior after nerve injury are associated with differential changes in brain-derived neurotrophic factor levels in mice subjected to early life stress[END_REF] in NMS animals. Moreover, OT analgesic action on thermal sensitivity also relies on an action on TRPV1 receptors, since OT seems able to directly activate and promote the rapid desensitization of TRPV1 receptors expressed by C-type sensory fibers [START_REF] Nersesyan | Oxytocin Modulates Nociception as an Agonist of Pain-Sensing TRPV1[END_REF].

Although not tested in this study, we cannot exclude such an effect in NMS animals, as an increased duration of thermal hot hyperalgesia could be due to the loss of OT action on TRPV1 receptors.

Nociceptive hypersensitivity could be easily explained by a long-lasting up-regulation of voltagegated channels Nav1.8 and Nav1.9 in DRG neurons as recently published [START_REF] Juif | Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation[END_REF].

In this study, impairment of the oxytocinergic pain control has been revealed after recruitment by a non-painful or painful stress. The hypothalamus, where OT producing neurons are located, seems particularly vulnerable to ELS and a dysfunction of the oxytocinergic system may contribute to these alterations as OT plays a key role in the modulation of stress responses, anxiety, social interactions and pain responses. Despite several trials, we have not been able to demonstrate that endogenous OT concentrations are different between groups. Recently, NMS in rodents was shown to be associated with changes in OTR and V1aR binding sites in specific brain regions [START_REF] Lukas | Maternal separation interferes with developmental changes in brain vasopressin and oxytocin receptor binding in male rats[END_REF], suggesting a vulnerability of this system to the early environment. In humans, ELS has been associated with low oxytocin concentrations in CSF [START_REF] Heim | Lower CSF oxytocin concentrations in women with a history of childhood abuse[END_REF]. Here, we show that OT analgesia is impaired after NMS, both in painful and non-painful conditions. To exert its analgesic effects at the spinal level, OT relays on three main mechanisms: (i) activation of a spinal microcircuit leading to an increased GABAergic inhibitory tone [START_REF] Breton | Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition[END_REF], (ii) decrease of the excitability of SC neurons in the superficial layers [START_REF] Breton | Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord[END_REF] and (iii) stimulation of allopregnanolone synthesis which further amplifies GABAergic inhibition by allosteric modulation [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF]. Here, we showed that spinal OTR are functional in NMS rats and that OTR activation can induce an efficient and transient analgesia, suggesting that the lack of OT-analgesia could hence result from supraspinal alterations. In agreement with our previously published results [22; 2], we failed to reveal any spinal contribution of the vasopressin V1A receptor in the fine tuning of basal nociceptive threshold or to induce oxytocinergic analgesia after painful or non-painful stress. This control was important since some discrepancies exist in the literature, often pointing out a specie-specific issue and a concentrationdependent action difficult to compare due to different administration procedures ( [START_REF] Schorscher-Petcu | Oxytocin-induced analgesia and scratching are mediated by the vasopressin-1A receptor in the mouse[END_REF]). Exploring hypothalamic OT neurons pharmacological and electrophysiological characteristics might help us to have a deeper understanding of the mechanisms leading to pain-related ELS consequences. A lack of AP synthesis following OTR activation could also explain the lack of OT anti-nociception following carrageenan inflammation in NMS rats, although we found no change in the spinal expression of transcripts coding for proteins responsible for AP synthesis in NMS rats, nor in the spinal expression of OTase mRNA.

Rescuing NMS long-term alterations of the nociceptive system

In our working hypothesis, we asked whether the lack of physical interaction between the mother and the pups could be a critical determinant to explain the deleterious plasticity of the nociceptive system and the deficit in oxytocinergic analgesia. Indeed, mother presence has already been shown to be protective against pain. Kangaroo mother care, in particular, is more frequently recommended and has been shown to have immediate analgesic effect on pain responses in the newborn [49; 50] and to be protective against long term alterations usually following preterm birth [51; 52]. As OT has a key role in the modulation of parental behavior and attachment [53; 54], we performed a rescue experiment by repeated neonatal OT treatment during NMS, which reversed some consequences of NMS on pain behavior. In the literature, OT has been proposed as a therapeutic agent for different neurodevelopmental pathologies such as autism spectrum disorders, since it decreases anxiety and optimizes social behavior [7; 8]. In animal models, postnatal injections of OT have been shown to prevent feeding, social and memory deficit in the Prader-Willi model [55; 29]. Here, we only studied the beneficial consequences of an early treatment with OT on pain behaviors but further experiments would be helpful to confirm if this treatment could also counteract the detrimental effects of NMS on pain-related comorbidities such as stress and anxiety. These observations might have great clinical relevance in neonatal intensive care units since it confirms, once again, the crucial role of physical interactions between the parents and the newborn to optimize newborn development and install proper homeostatic controls to cope with stress (including pain).

A close reciprocal relationship exists between the oxytocinergic system and neurosteroids.

Allopregnanolone is not only a downstream second messenger of OTR activation [START_REF] Juif | Long-lasting spinal oxytocin analgesia is ensured by the stimulation of allopregnanolone synthesis which potentiates GABA(A) receptor-mediated synaptic inhibition[END_REF], it has wellknown anxiolytic, analgesic and neuroprotective properties [START_REF] Poisbeau | Analgesic strategies aimed at stimulating the endogenous production of allopregnanolone[END_REF]. In this study, a rescue experiment with neonatal allopregnanolone treatment appeared successful to restore normal pain sensitivity and a functional OT analgesia after FSS. Neonatal allopregnanolone level alterations can indeed lead to changes in behavior at adulthood [START_REF] Darbra | Neonatal allopregnanolone levels alteration: effects on behavior and role of the hippocampus[END_REF]. Our result is also in good agreement with other studies using AP treatment to prevent neonatal anxiety after a short-term NMS (24 hours) [START_REF] Llido | Interaction between neonatal allopregnanolone administration and early maternal separation: effects on adolescent and adult behaviors in male rat[END_REF].

The mechanisms by which AP could be protective against NMS consequences on pain controls are still unknown. However, neonatal AP level alterations could interact with chloride homeostasis development, since AP has also been shown to change KCC2 expression in the hippocampus [START_REF] Modol | Neonatal allopregnanolone or finasteride administration modifies hippocampal K(+) Cl(-) co-transporter expression during early development in male rats[END_REF].

Eventually, it is not surprising that HDAC inhibitor and epigenetic changes are involved in the longterm consequences of NMS on nociception and OT analgesia. It has long been known that neonatal stress has long lasting effects through epigenetic mechanisms [32; 59; 33]. Epigenetic changes in chromatin compaction through histone modification such as histone acetylation have been demonstrated after NMS in different brain areas [59; 60; 19]. Here we also demonstrate that the spinal expression of different pain-related epigenetic factors is altered after NMS, in particular HDAC 1, 7 and 8 which are overexpressed in a long-lasting manner compatible with the observed pain phenotypes. In the pain context, histone acetylation can modulate nociceptive response in inflammatory neuropathic and visceral pain models [START_REF] Wang | Is there any therapeutic value for the use of histone deacetylase inhibitors for chronic pain?[END_REF]. Spinal class II HDACs are involved in inflammatory hyperalgesia [START_REF] Bai | Inhibition of class II histone deacetylases in the spinal cord attenuates inflammatory hyperalgesia[END_REF], which could explain the beneficial effects of SAHA treatment observed in this study after carrageenan inflammation, and the critical role of spinal HDAC 7 overexpression in NMS animals. In line, it has been suggested that suppressing HDAC 1 expression in the SC attenuated neuropathic pain symptoms [START_REF] Cherng | Baicalin ameliorates neuropathic pain by suppressing HDAC1 expression in the spinal cord of spinal nerve ligation rats[END_REF]. The OTR gene promoter is also subjected to increased association of acH3K14 after NMS in rats, in critical structures for pain control [START_REF] Litvin | Maternal separation renders adult rats hypersensitive to noxious stimuli and incapable to recruit oxytocin analgesic controls. Rescue is achieved by neonatal treatments with oxytocin, allopregnanolone or HDAC inhibitors[END_REF]. However, we did not see any difference in the spinal expression of OTR in our study, providing further evidence that histone modification may be necessary but not sufficient to exert significant and long lasting changes in specific gene expression and that epigenetic alterations targeting the OTR gene may actually be region-dependant.

Corroborating our results, Moloney and colleagues showed that NMS is associated with an alteration in histone acetylation at the spinal level [START_REF] Moloney | Early-life stress-induced visceral hypersensitivity and anxiety behavior is reversed by histone deacetylase inhibition[END_REF] and were the first to show that adult SAHA treatment can reverse NMS-induced visceral hypersensitivity. Here, we show that a preventive neonatal treatment with the same agent is also effective to prevent mechanical and thermal hypersensitivity at adulthood. At this stage, the underlying mechanisms are still to be identified as well as the molecular targets by which SAHA exerts its protective effects against NMS.

In conclusion, NMS induced both a basal hypersensitivity to pain and a dysfunction in OT descending control of pain, in painful and non-painful conditions. Some of these alterations can be rescued by neonatal SAHA or OT/AP treatment, but other alternatives need to be explored to be able to recover completely the OT antinociceptive action under inflammatory conditions. By studying the mechanisms of action of these pharmacological treatments, we might be able to identify new therapeutic targets to avoid NMS induced reprogramming of pain circuits. Carrageenan injection was done at day 0 just after the daily measure. Note that the duration of thermal hyperalgesia was longer for NMS rats. Statistical code for repeated measures Two Way ANOVA treatment X time followed by Sidak post hoc test to compare ipsilateral and contralateral hindpaws for each time point: **p<0.01, ***p<0.001 N=12 per group.

FIGURES AND LEGENDS

  ± 7.1 g, N = 12; p < 0.001, unpaired Student t-test, t = 18.83, df = 22) and paw withdrawal latencies to noxious heat (control: 21.1 ± 0.8 s, N = 12; NMS: 12.6 ± 0.4 s, N = 12; p < 0.001, unpaired Student t-test, t = 9, df = 22).

  injection: 145.75 ± 10.3 g, N = 8, Mann-Whitney test: U = 20.0; p = 0.2241) nor thermal nociceptive thresholds (before: 6.13 ± 0.4 s; after i.t. injection: 5.5 ± 0.5 s, N=8; Mann-Whitney test: U = 22.0; p = 0.3514) in NMS rat.

Figure 1 .

 1 Figure 1. Altered baseline nociceptive sensitivity and stress induced analgesia efficacy in NMS

Figure 2 .

 2 Figure 2. Increased sensibility to inflammatory pain in NMS rats. Time course of mechanical (top

Figure 3 .

 3 Figure 3. Lack of efficiency of OT anti-hyperalgesic control on behavioral symptoms of

Figure 4 .

 4 Figure 4. Lack of efficiency of OT anti-nociceptive control on spinal cord wide dynamic range

Figure 5 .

 5 Figure 5. Function of OTR in NMS rats. A. Expression of transcripts coding for spinal OTR in control

Figure 6 .

 6 Figure 6. Neonatal treatment with OT or AP can partially suppress NMS consequences. Effect of

Figure 7 .

 7 Figure 7. Epigenetic alterations following NMS and partial suppression of NMS consequences

Acknowledgments

This project has been supported by public funding from University of Strasbourg and CNRS. MM and GG are fellows of the French Ministry of research. PEJ was awarded a Gisele Guilbaud/Sfetd research price in 2013.

Conflict of interest statement

The authors have no conflict of interest. qPCR. No significant difference between conditions has been detected using One Way ANOVA, N=8 CTRL and NMS. Supplementary Table 1. List of primer sequences used to amplify genes of interest by quantitative RT-PCR.

Supplementary Figure1

Click here to download Supplementary Materials: figures, tables Suppl Figure 1.tif Supplementary Table1

Click here to download Supplementary Materials: figures, tables Suppl Table 1.tif


Pierrick Poisbeau

Valery Grinevich

Alexandre Charlet
email: acharlet@unistra.fr

Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels

Keywords: Amygdala, Hypothalamus, Nociception, Oxytocin, Oxytocin receptor, Pain, Spinal cord

Originally confined to the initiation of parturition and milk ejection after birth, the hypothalamic nonapeptide oxytocin (OT) is now recognized as a critical determinant of social behavior and emotional processing. It accounts for the modulation of sensory processing and pain perception as OT displays a potent analgesic effect mediated by OT receptors (OTRs) expressed in the peripheral and central nervous systems. In our chapter, we will first systemically analyze known efferent and afferent OT neuron projections, which form the anatomical basis for OT modulation of somatosensory and pain processing. Next, we will focus on the synergy of distinct types of OT neurons (e.g., magno-and parvocellular OT neurons) which efficiently control acute inflammatory pain perception. Finally, we will describe how OT signaling mechanisms in the spinal cord control nociception, as well as how OT is able to modulate emotional pain processing within the central amygdala. In the conclusions at the end of the chapter, we will formulate perspectives in the study of OT effects on pain anticipation and pain memory,

Introduction

Since the synthesis of oxytocin (OT) by the groups of Vincent du Vigneaud (du Vigneaud 1954(du Vigneaud -1955) ) and Roger Acher [START_REF] Acher | Chemistry of posterior-pituitary hormones[END_REF], OT has been classically described as a neurohormone, synthesized exclusively in the hypothalamic nuclei. OT binds to a single type of OTR [START_REF] Kimura | Structure and expression of a human oxytocin receptor[END_REF], expressed by myometrial cells of the uterine and myoepithelial cells of the mammary gland. Muscle contraction is then ensured by an intracellular Ca 2+ increase, triggered by the local production of inositol trisphosphate thanks to a G q protein coupling to phospholipase C.

In addition to its neurohormonal action on peripheral targets, OT acts as a neuromodulator in the central nervous system (CNS) and modulates a large number of brain functions, including social behaviors, emotions, and pain [START_REF] Lee | Oxytocin: the great facilitator of life[END_REF]; see also [START_REF] Bosch | Oxytocin and social relationships: from attachment to bond disruption[END_REF]. Despite the diverse behavioral effects of OT, we only start to understand the complexity of OTR-mediated intra-neuronal signaling (see [START_REF] Busnelli | Molecular basis of oxytocin receptor signalling in the brain: what we know and what we need to know[END_REF]. OTR are G-protein-coupled receptors which are capable of either increasing [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF][START_REF] Stoop | Neuromodulation by oxytocin and vasopressin[END_REF] or inhibiting [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF] neuronal excitability due to different mechanisms. One of these mechanisms is the dual OTR coupling to G q or G o /G i proteins [START_REF] Gravati | Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor[END_REF][START_REF] Busnelli | Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes[END_REF].

However, little is known about the intracellular OTR effectors and signaling pathways downstream of G protein activation and, in particular, about the effectors known to be relevant to neuronal cells and brain functions. One of the most important intracellular signaling pathways activated by OTRs is the mitogen-activated protein kinase (MAPK) cascade. Inga Neumann's team has demonstrated that OTR/MEK/ ERK signaling mediates OT anxiolytic effects [START_REF] Blume | Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus[END_REF][START_REF] Jurek | Differential contribution of hypothalamic MAPK activity to anxiety-like behaviour in virgin and lactating rats[END_REF][START_REF] Van Den Burg | Oxytocin stimulates extracellular Ca2+ influx through TRPV2 channels in hypothalamic neurons to exert its anxiolytic effects[END_REF], which requires the influx of extracellular calcium through transient receptor potential vanilloid (TRPV) channels. In particular, OTR activation in hypothalamic neurons induces the release of G bg and PI3K activation, thus promoting the incorporation of TRPV channels in the plasma membrane and consequent calcium influx and MEK 1/2 phosphorylation (van den [START_REF] Van Den Burg | Oxytocin stimulates extracellular Ca2+ influx through TRPV2 channels in hypothalamic neurons to exert its anxiolytic effects[END_REF]. It is likely that Ca 2+ -activated calmodulin-dependent activation of the EGFR is involved in this process, but the cascade of the molecular players involved is still unknown [START_REF] Blume | Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus[END_REF]. It is interesting to note that OTR activation has also been associated with the stimulation of ERK 1/2 activity in the superficial layers of the spinal cord, as well as the local increased production of analgesic neurosteroids (Juif et al. 2013). It is expected that OTRs activate other signaling pathways in neuronal (and glial) cells and progress in this area will certainly contribute to further advances in OT studies.

In particular, it would not be surprising to observe particular signaling cascades as a function of the brain-region function, including those involved in pain processing. Indeed, OT axonal projection and OTR expression were shown in several structures known to process the different components of "pain messages," such as the superficial layers of spinal cord, the medial amygdala, the periaqueductal gray matter, or the ventral tegmental area (Fig. 1).

As illustrated in Fig. 1, pain processing involves a large number of CNS structures and gives rise to an unpleasant emotion which can be defined as "an adaptive aversive experience with sensory, emotional, cognitive, and social components" [START_REF] Williams | Updating the definition of pain[END_REF]. To become a conscious sensation, nociceptive messages reach the cortical areas using various ascending tracts (spinothalamic, spinoreticular, and spino-parabrachio-amygdaloid tracts). Meanwhile, collaterals are sent to intermediate structures in the brainstem (rostroventral medulla and periaqueductal gray), the limbic system (amygdala), and the diencephalon (hypothalamus and thalamus). Processing of nociceptive messages by these structures is accompanied by the parallel recruitment of descending pain controls located in some cortical areas, hypothalamus, amygdala, and the brainstem. OT neurons are involved in this active process at hierarchically different levels, described in the section below.

Physiological and Behavioral Evidence

The role of OT in pain modulation has long been controversial. Although the doses and administration methods used could have been different in the available literature, an anti-nociceptive effect of OT has been clearly demonstrated in healthy rats [START_REF] Arletti | Influence of oxytocin on nociception and morphine antinociception[END_REF][START_REF] Lundeberg | Anti-nociceptive effects of oxytocin in rats and mice[END_REF][START_REF] Agren | The oxytocin antagonist 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin reverses the increase in the withdrawal response latency to thermal, but not mechanical nociceptive stimuli following oxytocin administration or massagelike stroking in rats[END_REF][START_REF] Petersson | Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats[END_REF]Juif and Poisbeau 2013) and in rodent models of inflammatory [START_REF] Petersson | Oxytocin decreases carrageenan induced inflammation in rats[END_REF][START_REF] Yu | Involvement of oxytocin in spinal antinociception in rats with inflammation[END_REF]Juif et al. 2013) or neuropathic pain [START_REF] Condes-Lara | Oxytocin actions on afferent evoked spinal cord neuronal activities in neuropathic but not in normal rats[END_REF][START_REF] Miranda-Cardenas | Oxytocin and electrical stimulation of the paraventricular hypothalamic nucleus produce antinociceptive effects that are reversed by an oxytocin antagonist[END_REF].

In healthy rat, systemic (i.p. or s.c.) or central injections (i.c.v. or intracisternally) of OT increases the nociceptive thresholds to observe an aversive behavior when rodents were submitted to noxious thermal or mechanical stimulation. In some studies, these effects seemed to involve the endogenous opioid system as OT antinociception was completely or partially blocked by the administration of on the right describes the cortical amygdala and thalamic regions of rodents innervated by OT fibers and the corresponding expression of OT receptors, if available. Text in red indicates that OT receptor expression has been confirmed in human samples. LC locus coeruleus, PAG periaqueducal gray, PBN parabrachial nucleus, RVM rostroventral medulla, SRD subnuclear reticularis dorsalis, HDB horizontal limb of the diagonal band of Broca antagonists of opioid receptors [START_REF] Arletti | Influence of oxytocin on nociception and morphine antinociception[END_REF][START_REF] Petersson | Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats[END_REF][START_REF] Yu | Involvement of oxytocin in spinal antinociception in rats with inflammation[END_REF]. Recently, an antinociceptive effect of OT has been characterized after i.v. injection of OT in concentrations close to physiological (Juif and Poisbeau 2013).

The anti-nociceptive effect of OT was not only observed after application of exogenous OT. Several studies in rodents suggested that the OT system could be considered as a "homeostatic" inhibitory control limiting the transmission of nociceptive messages [START_REF] Agren | The oxytocin antagonist 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin reverses the increase in the withdrawal response latency to thermal, but not mechanical nociceptive stimuli following oxytocin administration or massagelike stroking in rats[END_REF][START_REF] Lund | Repeated massage-like stimulation induces long-term effects on nociception: contribution of oxytocinergic mechanisms[END_REF][START_REF] Robinson | Oxytocin mediates stress-induced analgesia in adult mice[END_REF]. For example, electrical stimulation of the PVN significantly reduced pain symptoms in neuropathic rats [START_REF] Miranda-Cardenas | Oxytocin and electrical stimulation of the paraventricular hypothalamic nucleus produce antinociceptive effects that are reversed by an oxytocin antagonist[END_REF]). Using optogenetic stimulation of spinal cord projecting parvOT neurons, a selective reduction in the nociceptive action potential discharge was also observed in deep dorsal horn neurons [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF]. Finally, OT analgesia may also be obtained after stress, when OT concentration increases in the blood [START_REF] Robinson | Oxytocin mediates stress-induced analgesia in adult mice[END_REF]Juif and Poisbeau 2013).

OT Neurons Involved in the Modulation of Pain

Magnocellular OT Neurons

The central OT system in humans consists of about 35,000 neurons [START_REF] Morton | A quantitative analysis of the normal neuron population of the hypothalamic magnocellular nuclei in man and of their projections to the neurohypophysis[END_REF][START_REF] Wierda | Oxytocin cell number in the human paraventricular nucleus remains constant with aging and in Alzheimer's disease[END_REF], corresponding to around 8,000 neurons in rats [START_REF] Rhodes | Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin[END_REF]. Most OT producing neurons are located in the paraventricular (PVN), supraoptic (SON), and accessory nuclei and are large cells (20-30 mm in diameter). They are referred to as "magnocellular neurons" (magnOT; Fig. 2) and project to the posterior pituitary through which they release OT into the systemic blood circulation [START_REF] Armstrong | Morphological and electrophysiological classification of hypothalamic supraoptic neurons[END_REF][START_REF] Burbach | Gene regulation in the magnocellular hypothalamo-neurohypophysial system[END_REF]. Over decades, the magnOT neurons were the main focus of researchers as the main source of plasma OT, which induces constriction of smooth muscle cells in uterus and myoepithelial cells in mammary glands to elicit labor and milk let-down, respectively [START_REF] Leng | 60 years of neuroendocrinology: the posterior pituitary, from Geoffrey Harris to our present understanding[END_REF]. However, very recently it was demonstrated that magnOT neurons, in addition to their projections to the posterior pituitary, also send the collaterals to vast majority forebrain regions to modulate brain-region specific behavior as fear expression [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF]) and the processing of social olfactory signals [START_REF] Oettl | Oxytocin enhances social recognition by modulating cortical control of early olfactory processing[END_REF]. Furthermore, magnOT neurons project to various forebrain regions receiving somatosensory and pain signals.

Parvocellular OT Neurons

Although magnOT neurons remain the primary subject of studies, Sawchenko and Swanson in the early 1980s demonstrated the existence of another population of OT neurons in the PVN, smaller than magnOT neurons, with a different shape of cell body and -most importantly -with projections to the spinal cord and several brain stem nuclei instead of projections to the posterior pituitary [START_REF] Sawchenko | Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat[END_REF]). These cells were named "parvocellular or pre-autonomic neurons" (parvOT; Fig. 2), but were only sporadically studied due to their small number (total number is not counted so far) and difficulties with their identification, as they do not occupy a clearly boarded position in the PVN, but intermingle with other cell types [START_REF] Swanson | Hypothalamic integration: organization of the paraventricular and supraoptic nuclei[END_REF]. However, studies on identified parvOT neurons have revealed that they are distinct not only in morphology and projection trajectories, but also in their electrophysiological characteristics: After injection of depolarizing currents in whole cell patch clamp configuration, parvOT neurons do not show the transient outward rectification that is typical for magnOT neurons [START_REF] Luther | Neurosecretory and non-neurosecretory parvocellular neurones of the hypothalamic paraventricular nucleus express distinct electrophysiological properties[END_REF][START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF].

Based on specific projections of parvOT neurons to the brainstem (rostro-ventral medulla, nucleus tractus solitarius, dorsal vagal complex, and nucleus phrenicus) and spinal cord (intermediolateral cell column), several reports have demonstrated their contribution to the regulation of autonomic functions that modulate cardiovascular responses, respiration, and gastric motility [START_REF] Geerling | Paraventricular hypothalamic nucleus: axonal projections to the brainstem[END_REF]). These different effects suggested an anatomical and functional diversity of OT neurons.

Most recently, we indeed demonstrated the existence of a specific subpopulation of about 30 parvOT neurons that project from the PVN to magnOT neurons in the ipsilateral and contralateral SON and, in addition, send axonal collaterals to sensory neurons of the spinal cord [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF]. The optogenetic stimulation of the axons of these parvOT within the SON elicited firing of magnOT neurons and caused a robust increase of OT release into the blood (van den [START_REF] Van Den Burg | Oxytocin stimulates extracellular Ca2+ influx through TRPV2 channels in hypothalamic neurons to exert its anxiolytic effects[END_REF]. This suggested a role of this particular group of parvOT neurons in the coordination of activity of magnOT neurons in various situations, including modulation of pain perception.

Circuits for Pain-Induced Activation of OT Neurons

Although it was reported that somatic and visceral pain activates OT neurons [START_REF] Ceccatelli | Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress[END_REF][START_REF] Wang | Proximal colon distension induces Fos expression in oxytocin-, vasopressin-, CRF-and catecholamines-containing neurons in rat brain[END_REF]; see also Fig. 3), there is very limited information on sensory projections onto OT neurons. Classical tracing studies with conventional anterograde tracer showed that neurons of the deep (II-X), but not superficial (I), layers of the spinal cord project to the caudo-dorsal part of the PVN [START_REF] Poisbeau | A comparative reappraisal of projections from the superficial laminae of the dorsal horn in the rat: the forebrain[END_REF], where parvOT neurons are located (Eliava et al. 3 OT neurons are activated by an acute pain. Left: merge; middle: c-fos (green); right: oxytocin (red). A repeated acute pain stimulation (tail immersion in 46 C for 30s, three times, under isoflurane anesthesia) activates a subset of OT neurons, suggesting that only a subpopulation of OT neurons may react to a nociceptive stimulus. This raise the question of the involvement of this particular nociceptive-sensible OT neurons in pain processing. Unpublished materials from Charlet and Grinevich 2016). In conjunction, two other anatomical studies have shown that the injection of wild-type rabies virus into the rat nipple resulted in the back-labeling of presumably parvOT neurons in the PVN [START_REF] Gerendai | Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique[END_REF][START_REF] Koves | Characterization of the autonomic innervation of mammary gland in lactating rats studied by retrograde transynaptic virus labeling and immunohistochemistry[END_REF]). However, the use of multi-synaptically spreading rabies virus has precluded assessing whether spinal cord neurons that receive sensory information from nipples indeed directly terminate on parvOT neurons or whether they project indirectly [e.g., via brainstem nuclei, such as the nucleus tractus solitarius [START_REF] Affleck | The projection and synaptic organisation of NTS afferent connections with presympathetic neurons, GABA and nNOS neurons in the paraventricular nucleus of the hypothalamus[END_REF]]. At the same time, no direct projections to OT neurons from amygdalar, thalamic, and cortical regions, associated with somatosensation/nociception, have been reported. As well, there is no information about OT cell types (i.e. ParvOT vs. MagnOT neurons) predominantly activated by pain.

Supraspinal Structures Involved in Oxytocinergic Modulation of Pain

Pain is a ubiquitous experience in animals, described by the International Association for the Study of Pain (IASP) as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage." The pain pathways consist of an ascending one, which convey the nociceptive signals from the periphery to the central nervous system, and a descending pathway, which projects onto different elements of the pathways to either increase or decrease in nociception. The ensembles of involved structures are called the "pain matrix," which is affected by OT by combined peripheral and central mechanisms [START_REF] Poisbeau | Oxytocin and the modulation of pain experience: implications for chronic pain management[END_REF].

A decade ago, it was shown that central (i.c.v.) injection of OT can alleviate pain [START_REF] Yang | Central oxytocin enhances antinociception in the rat[END_REF]. More recently, it was reported that OT axons innervate structures of the "pain matrix" (Fig. 1) and OT was shown to have a pain-related effect via PAG [START_REF] Ge | Blockade effect of mu and kappa opioid antagonists on the antinociception induced by intra-periaqueductal grey injection of oxytocin in rats[END_REF], nucleus raphe magnus [START_REF] Wang | Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of mu-opioid receptor[END_REF], nucleus accumbens [START_REF] Gu | Involvement of opioid receptors in oxytocin-induced antinociception in the nucleus accumbens of rats[END_REF], and central amygdala [START_REF] Han | Involvement of oxytocin and its receptor in nociceptive modulation in the central nucleus of amygdala of rats[END_REF], while few evidences obtained in humans show oxytocin pain-related effects in the cortical areas [START_REF] Blume | Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus[END_REF].

Cortex

Functional neuroimaging approaches in humans show a robust activation in the cortical structures of the pain matrix, mainly insula and sensorimotor regions, when subjects were asked to observe pain in others. As this activation was strongly reduced after intranasal OT, the authors conclude that OT may decrease empathy for pain [START_REF] Blume | Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus[END_REF]. However, while OT fibers are highly present in a number of pain-related cortical regions [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF]; Fig. 1) their putative analgesic role is still to be deciphered.

Central Amygdala

The central amygdala is a key component in the pain matrix that assigns emotional to salient external stimuli [START_REF] Sah | The amygdaloid complex: anatomy and physiology[END_REF] and is thus a key structure in the regulation of pain and its associated mood alterations [START_REF] Neugebauer | Forebrain pain mechanisms[END_REF]. Viviani and colleagues showed that OT responsive CeL interneurons can selectively gate different responses (to fear in this case) by acting on a subpopulation of projection neurons of the CeM [START_REF] Viviani | Oxytocin selectively gates fear responses through distinct outputs from the central amygdala[END_REF]. The physiological relevance of such regulation of CeA by OT was further tested. Using optogenetics to allow specific activation of the axons of OT neurons, Knobloch and colleagues showed that activation of OT axons in the CeL elicited the same responses in CeA GABAergic neurons as did exogenous applications, but revealed a further glutamatergic component in this response [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF]. In a contextual fear conditioning paradigm, they could reduce freezing by optogenetic activation of OT axons in the CeL, demonstrating that the sparse OT fibers innervating the CeL are sufficient to elicit a drastic change in behavior in the rat [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF]. Because of the strong link between pain and anxiety, as well as the known involvement of CeA in pain modulation, one can speculate about the important and complex analgesic function of OT release in this structure. Accordingly, Han and Yu demonstrated an analgesic effect of intra-CeA infusion of oxytocin [START_REF] Han | Involvement of oxytocin and its receptor in nociceptive modulation in the central nucleus of amygdala of rats[END_REF]). In the same supportive line of evidence, Neugebauer's lab recently published interesting results suggesting that the CeA OTR mediates some analgesic tone in an arthrisis pain model [START_REF] Cragg | Differential contributions of vasopressin V1A and oxytocin receptors in the amygdala to pain-related behaviors in rats[END_REF].

PAG

The PAG and the rostral ventromedial medulla are highly connected and are key centers of descending pain pathways [START_REF] Heinricher | Descending control of nociception: specificity, recruitment and plasticity[END_REF]). It has been shown that painful stimulations are able to elevate the OT content in the PAG (Yang et al. 2011a). In addition, intra-PAG injection of OT can increase mechanical nociceptive threshold in naı ¨ve rats (Yang et al. 2011b). However, as oxytocin at the concentrations used can bind both OTR and V1A-R, further experiments have to be conducted to determine both the involvement of OTR itself as well as of endogenous oxytocin in pain control through modification of PAG activity. Finally, the precise circuitry involved in such an antinociceptive effect is currently unknown, as are the potential analgesic effects of oxytocin in a painful situation.

Raphe Nuclei

Raphe magnus has been reported as a potent target of the OT-mediated analgesic system, as direct infusion within the raphe magnus induces strong analgesia [START_REF] Wang | Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of mu-opioid receptor[END_REF]. Interestingly, the serotoninergic system is also involved in OT pain modulation and indeed the raphe nuclei shows increase in c-fos after PVN stimulation known to induce anti-nociception in SC [START_REF] Condes-Lara | Hypothalamic paraventricular nucleus stimulation enhances c-Fos expression in spinal and supraspinal structures related to pain modulation[END_REF]. Serotonin intrathecal injection mimics the OT effect and can potentiate its anti/nociceptive effect if administrated together. In addition, OT injection in the raphe magnus also reduces anxiety [START_REF] Yoshida | Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice[END_REF]. This is of particular interest because pain is a complex feeling, involving both somatosensory and emotional components. In humans, intranasal OT administration resulted in a decrease of anxiety and improved trust [START_REF] Heinrichs | Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress[END_REF][START_REF] Kirsch | Oxytocin modulates neural circuitry for social cognition and fear in humans[END_REF]. As chronic pain is linked to the development of anxio-depressive symptoms [START_REF] Asmundson | Understanding the co-occurrence of anxiety disorders and chronic pain: state-of-the-art[END_REF] and knowing the various effects of central and peripheral OT on both, some crucial therapies might emerge through a better comprehension of the OT system in the CNS.

Nucleus Accumbens

A striking example of the dramatic role induced by a subtle change in OT signaling is well illustrated in the nucleus accumbens. Indeed, changes of OTR expression on reproductive/social behavior drives the variation in OTR expression in the nucleus accumbens of voles, making them either monogamous (prairie voles, more OT-binding) or polygamous (mountain voles, less OT-binding) [START_REF] Insel | Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles[END_REF]. Interestingly, Gu and Yu suggested a dose-dependent antinociceptive action of OT when directly infused in the nucleus accumbens [START_REF] Gu | Involvement of opioid receptors in oxytocin-induced antinociception in the nucleus accumbens of rats[END_REF]. While this mechanism of action is still unclear regarding which receptor it activates or which pathways it recruits, this data is very interesting in the view of the physiological and rhythmic variability of OTR expression, as exemplified by Insel and Shapiro.

Spinal Structures Involved in Oxytocinergic Modulation of Nociception

Oxytocinergic Modulation of Spinal Network

The spinal cord is one of the CNS regions where there is a perfect overlap between the oxytocinergic axonal innervation and OT binding sites (Fig. 4). ParvOT project their axons in areas of the spinal cord known to process nociceptive and non-nociceptive somatic (layer I, II) and visceral informations (layer X, spinal lateral nucleus). Projection areas are also located in the intermediate spinal cord gray matter, i.e., specifically those processing sympathetic (intermedio-lateral columns (ilc) and intermedio-median autonomous area (ima) around the central canal) and parasympathetic autonomic formations (e.g., sacral parasympathetic nucleus). As illustrated in Fig. 4, these areas perfectly overlap with OT binding sites detected by histoautoradiography [START_REF] Reiter | Localization of oxytocin binding sites in the thoracic and upper lumbar spinal cord of the adult and postnatal rat: a histoautoradiographic study[END_REF][START_REF] Veronneau-Longueville | Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat[END_REF][START_REF] Uhl-Bronner | Sexually dimorphic expression of oxytocin binding sites in forebrain and spinal cord of the rat[END_REF].

OT innervation in rats is particularly abundant around the thoracic and lumbar segments. In agreement, radioimmunoassay revealed that OT spinal content was also significantly higher in these particular spinal segments (Juif et al. 2013). Finally, the density of OT binding in superficial layers I and II of the lumbar segments was significantly higher in L4 and L6 compared to the others, when quantified by autoradiography. Together, these data provide an anatomical support for a direct modulation of spinal cord neurons by parvOT neurons, possibly affecting nociceptive and autonomic processing. It is interesting to note that OTergic synapses in the superficial layers could be visualized in electron microscopy (Rousselot ), although no information was given on the neurochemical nature of the postsynaptic neurons. Electrophysiological experiments revealed that OT-sensitive neurons could be of glutamatergic nature. A presynaptic excitation of glutamatergic transmission was found after infusion of OT in primary cultures of layer I-III rat spinal cord neurons [START_REF] Jo | Oxytocin modulates glutamatergic synaptic transmission between cultured neonatal spinal cord dorsal horn neurons[END_REF]. OT-activated spinal cord neurons, revealed by c-fos expression, were always non-GABAergic neurons in acute rat slices [START_REF] Breton | Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition[END_REF]. It was further shown that in lamina II, OTR activation reduces the firing of neurons exhibiting a depolarization-induced bursting firing pattern and has no effect on the single spike firing pattern cells [START_REF] Breton | Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord[END_REF]. Of importance, it seems that the AVP system is not involved in those effects of OT at the spinal level [START_REF] Rojas-Piloni | Oxytocin, but not vassopressin, modulates nociceptive responses in dorsal horn neurons[END_REF]. Those effects of exogenous OT can also be observed by stimulation of the descending OT projections from the PVN: while recording the nociceptive Aδ and C evoked discharge, Conde `s-Lara and colleagues demonstrated a reduction of the duration of such discharge by raw electrical stimulation of the PVN [START_REF] Condes-Lara | Paraventricular hypothalamic influences on spinal nociceptive processing[END_REF]; see also Fig. 5). In vivo studies by DeLaTorre and colleagues further showed that exogenous OT or endogenous OT released through PVN stimulation could reduce or prevent the LTP in spinal wide dynamic range neurons [START_REF] Delatorre | Paraventricular oxytocinergic hypothalamic prevention or interruption of long-term potentiation in dorsal horn nociceptive neurons: electrophysiological and behavioral evidence[END_REF]). The analgesic role of endogenous release of OT in the spinal cord was further demonstrated and extended to lamina X, imc and ima: about 30 parvOT neurons can both control magnOT-mediated OT blood release and directly target deep layer WDR neurons to ensure a coordinated and complementary effect of peripheral and central release of OT on nociception [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF]. Finally, these findings can be translated into human clinics as intrathecal OT injections successfully reduce both low-back [START_REF] Yang | Intrathecal administration of oxytocin induces analgesia in low back pain involving the endogenous opiate peptide system[END_REF]) and visceral [START_REF] Engle | Intrathecal oxytocin inhibits visceromotor reflex and spinal neuronal responses to noxious distention of the rat urinary bladder[END_REF] pain.

Interestingly, here the OT system seems to be in strong interaction with several others. For example, the long-lasting analgesia induced by OT is mediated by a strong neurosteroidogenesis, which leads to increase GABA A mediated inhibition in lamina II neurons (Juif et al. 2013). This model explains the transition from acute to chronic pain by potentiation (LTP) of nociceptive neurons after repeated noxious stimuli (or other mechanisms), which strengthen further the nociceptive signals. In addition, μ-opioid receptor seems to be involved in the OT-mediated analgesic effect as their blockade do partially block the effect of either OT application or PVN stimulation [START_REF] Miranda-Cardenas | Oxytocin and electrical stimulation of the paraventricular hypothalamic nucleus produce antinociceptive effects that are reversed by an oxytocin antagonist[END_REF][START_REF] Condes-Lara | Paraventricular hypothalamic oxytocinergic cells responding to noxious stimulation and projecting to the spinal dorsal horn represent a homeostatic analgesic mechanism[END_REF]). Similarly, another study showed the participation of mu and kappa opioid receptors in the antinociceptive effect of OT in an inflammatory pain model [START_REF] Yu | Involvement of oxytocin in spinal antinociception in rats with inflammation[END_REF].

Mechanisms for Spinal Antinociception

Neurons in the superficial layers of the dorsal horn spinal cord play a fundamental role in nociceptive processing, since they are the first relay for "nociceptive messages" transmitted by sensory afferent fibers type Aδ and C [START_REF] Poisbeau | Spinal cord mechanisms in acute and chronic pain states[END_REF]. They are also the target for descending controls including parvOT axonal terminals Unpublished materials from Juif and Poisbeau secreting OT. Using patch-clamp electrophysiology in spinal cord slice combined to c-fos expression, OT has been shown to excite a subpopulation of glutamatergic interneurons [START_REF] Breton | Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition[END_REF]. Interestingly, these OT-sensitive excitatory interneurons were apparently responsible for the recruitment of all GABAergic interneurons, thus inducing a general increase in inhibition in the superficial layers. In addition to a possible presynaptic inhibition on nociceptive primary afferent fibers, this mode of action of OT could be sufficient to explain the selective blockade of Aδ and C-type nociceptive messages. So far, it has been impossible to observe any calcium concentration increase in spinal cord neurons and the detailed mechanism of action is still not fully understood. In the short term, it seems that OTR activation can rapidly inhibit transient potassium current (I A ) and reduce the firing ability of some spinal cord neurons expressing this channel [START_REF] Breton | Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord[END_REF]. This reduction of neuronal firing is interesting since it will (1) contribute to the general decrease in interneuronal network ongoing activity without affecting the amplified inhibitory synaptic activity and, (2) reduce (or prevent) the integration of nociceptive messages by projection neurons located in lamina I and V (deep layers). This hypothesis has been indeed verified while recording neurokinin 1 receptor (NK1R) positive wide dynamic range neurons in lamina V, exposed to OT [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF]). These NK1R neurons in lamina V could even express OTR, as shown in this study.

A remaining open question was related to a possible long-lasting analgesic effect of OT. In inflammatory pain states, OT content in the spinal cord remained elevated for several days (Juif et al. 2013). In this study, a novel mechanism has been proposed where OTR activation exerts a tonic activation of extracellular regulated kinases type and stimulates the production of the neuroactive neurosteroid allopregnanolone. Local increase in allopregnanolone concentration perfectly explained the persistence of an elevated inhibition mediated by prolonged GABAA receptor-mediated synaptic currents and reduction in membrane resistance due to tonic extrasynaptic GABAA receptor activation.

Conclusion

After more than two decades of work, there is nowadays a large number of studies supporting OT analgesic action in the central nervous system. For practical reasons, and also because the spinal cord contains second order neurons receiving peripheral "pain messages," spinal antinociception has been well characterized. Apart from its sensory component (i.e., nociception), pain is an emotion giving rise to robust aversive behaviors and long-lasting cognitive imprinting. Recent progress has been made by analyzing the role of OT in CNS structures processing emotion such as fear and anxiety. The benefits of OT in limiting fear expression and anxiety is undeniable and may contribute, for a large part, to the limitation of pain expression in physiological and pathological conditions. It is, however, unclear how, to what extent, and on which component OT can modulate pain expression at this stage. In particular, the role of OT on rewarding processes and memory reinforcement remains to be unraveled. This question is obviously a critical issue when considering labor pain and possibly other visceral pain conditions which are particularly difficult to treat.

The clinical interest of using OT as a painkiller in pathological pain states has then to be considered. Two studies are already available and have been highly discussed. The first one attempted to alleviate pain symptoms in fibromyalgic female patients and was inconclusive at a daily administration dose of 80 IU for 3 weeks [START_REF] Mameli | Oxytocin nasal spray in fibromyalgic patients[END_REF]). In the second study, two patients suffering from refractory cancer pain reported to have less pain symptoms after epidural injection of about 2 μg of OT [START_REF] Condes-Lara | Successful pain management with epidural oxytocin[END_REF]. Of course, these two studies are too preliminary to conclude. The future human studies with larger cohorts, pertinent administration procedures, and appropriate design will, for sure, be of significant interest to propose (or not) OT treatment to pain patients.

Among the remaining open questions, a subject is now appearing: a possible analgesic role of OT at the periphery. Indeed, it appears that some small diameter sensory neurons (i.e., C-type nociceptors) do express OT receptor [START_REF] Moreno-Lopez | Identification of oxytocin receptor in the dorsal horn and nociceptive dorsal root ganglion neurons[END_REF], explaining nicely antinociception observed after intravenous OT injection (Juif and Poisbeau 2013) or optogenetic-driven endogenous release from SON [START_REF] Eliava | A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing[END_REF]. A similar conclusion was reached while studying the trigeminal ganglia in the context of migraine [START_REF] Tzabazis | Oxytocin receptor: expression in the trigeminal nociceptive system and potential role in the treatment of headache disorders[END_REF]. In addition to what has been already shown in the central nervous system, these recent results on peripheral OT receptor activation provide a great hope of therapeutic benefit for patients suffering from migraine and other primary headache disorders.

Fig. 1

 1 Fig. 1 Simplified diagram of major pain pathways and of key structures of the so-called "pain matrix." Ascending pathways are shown in red whereas descending pathways are shown in green. Structures highlighted have been shown to be innervated by OT fibers. Table on the right describes the cortical amygdala and thalamic regions of rodents innervated by OT fibers and the corresponding expression of OT receptors, if available. Text in red indicates that OT receptor expression has been confirmed in human samples. LC locus coeruleus, PAG periaqueducal gray, PBN parabrachial nucleus, RVM rostroventral medulla, SRD subnuclear reticularis dorsalis, HDB horizontal limb of the diagonal band of Broca

Fig. 2

 2 Fig. 2 Morphological and functional differences between magno-and parvocellular oxytocinergic neurons. (a) Parvocellular OT neuron. (b) Magnocellular neurons. Right traces: Current steps protocol starting from a hyperpolarizing current chosen to reach À100 mV (here 100 pA) followed by progressively more depolarizing current injections (upper trace). The representative changes in membrane potential for the parvOT and magnOT PVN neurons during the part of the current steps as indicated by the zoomed area are shown (lower traces). The ParvOT neurons (middle trace) do not display the transient outward rectification specific for the magnOT neurons (lower trace). From Eliava et al. (2016)

Fig.

  Fig.3OT neurons are activated by an acute pain. Left: merge; middle: c-fos (green); right: oxytocin (red). A repeated acute pain stimulation (tail immersion in 46 C for 30s, three times, under isoflurane anesthesia) activates a subset of OT neurons, suggesting that only a subpopulation of OT neurons may react to a nociceptive stimulus. This raise the question of the involvement of this particular nociceptive-sensible OT neurons in pain processing. Unpublished materials from Charlet and Grinevich

Fig. 4

 4 Fig. 4 Oxytocin in spinal cord. Immunohistochemistry of neurophysin I-(OT) immunoreactive axons in the spinal cord (left) and autoradiography of OT binding sites (right) using the radiolabeled selective antagonist dOVT-I 125 (right) illustrating the perfect overlap in the superficial layers (layer I-II), lateral corticospinal tractus (LCSp), intermedio-lateral (IML), and intermediomedial (IMM) nuclei and layer X. Adapted from Breton et al. (2008); unpublished materials from Charlet and Grinevich

Fig. 5

 5 Fig. 5 Representative example of a single unit extracellular recording of wide dynamic range spinal cord neurons in deep layers of the dorsal horn during repetitive electrical stimulation of hindpaw receptive field, before and after stimulation of paraventricular nuclei (PVN). In bottom insets are illustrated the cumulative number of action potentials recorded, originating from fast and slow conducting sensory afferent (A and C). Note that PVN stimulation specifically reduced Aδ and C discharges (and C-related postdischarges).

Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels

P.Poisbeau et al.