Muris Humo

Beyza Ayazgök

Léa J Becker

Elisabeth Waltisperger

Tomi Rantamäki

Ipek Yalcin
email: yalcin@inci-cnrs.unistra.fr

Ketamine induces rapid

Keywords: Ketamine, Neuropathic pain-induced depression, Comorbidity, Anterior cingulate cortex, MKP-1, pERK ACC, anterior cingulate cortex, ATF1, transcription factor 1, CREB, cyclic AMP (adenosine monophosphate) response element binding protein, ERK, extracellular signal regulated kinase, JNK, c-Jun N-terminal kinase, MAPK, mitogen activated protein kinase, MKP-1, MAPK phosphatase-1, NMDA, N-Methyl-d-Aspartate, pERK,

Introduction

Chronic pain and depression are detrimental conditions affecting an increasing number of people around the world [START_REF] Bair | Depression and pain comorbidity: a literature review[END_REF][START_REF] Rayner | Depression in patients with chronic pain attending a specialised pain treatment centre: prevalence and impact on health care costs[END_REF]. Moreover, the co-existence of these conditions represents a serious socio-economic burden and results in a more pronounced disability and a poorer prognosis than either condition alone [START_REF] Arnow | Comorbid depression, chronic pain, and disability in primary care[END_REF][START_REF] Gallagher | Managing pain and comorbid depression: A public health challenge[END_REF]. Preclinical data suggests that the comorbid relationship of chronic pain and depressive-like behaviors can be modeled in murine models [START_REF] Humo | The molecular neurobiology of chronic pain-induced depression[END_REF]Yalcin et al., 2014a), which allows studying molecular characteristics and treatment strategies in more depth.

Ketamine is a versatile pharmacological agent described in 1965 [START_REF] Domino | PHARMACOLOGIC EFFECTS OF CI-581, A NEW DISSOCIATIVE ANESTHETIC[END_REF] and extensively used in clinical practice since 1970 [START_REF] Aroni | Pharmacological aspects and potential new clinical applications of ketamine: reevaluation of an old drug[END_REF]. It primarily acts as a noncompetitive antagonist of the glutamatergic N-methyl-D-aspartate (NMDA) receptors [START_REF] Bergman | Ketamine: review of its pharmacology and its use in pediatric anesthesia[END_REF]. Although initially used as a dissociative anesthetic, ketamine has been later

shown efficient in treating both depression and pain [START_REF] Abdallah | Ketamine as a promising prototype for a new generation of rapid-acting antidepressants[END_REF][START_REF] Persson | Ketamine in pain management[END_REF]. However, while there is widespread evidence of ketamine use for the individual treatment of chronic pain and depression in humans (Aan Het Rot et al., 2012;[START_REF] Hocking | Ketamine in chronic pain management: an evidence-based review[END_REF], evidence for its beneficial use in the comorbidity of these two conditions is highly limited [START_REF] Bigman | Use of ketamine for acute suicidal ideation in a patient with chronic pain on prescribed cannabinoids[END_REF][START_REF] Weber | Case Report of Subanesthetic Intravenous Ketamine Infusion for the Treatment of Neuropathic Pain and Depression with Suicidal Features in a Pediatric Patient[END_REF]. Moreover, despite the increasing number of evidence accumulated over the past several decades about the antidepressant and antinociceptive activity of ketamine [START_REF] Cohen | Consensus Guidelines on the Use of Intravenous Ketamine Infusions for Chronic Pain From the American Society of Regional Anesthesia and Pain Medicine, the American Academy of Pain Medicine, and the American Society of Anesthesiologists[END_REF][START_REF] Sleigh | Ketamine -More mechanisms of action than just NMDA blockade[END_REF]Zanos and Gould, 2018a, b), the underlying mechanism of ketamine action in the chronic pain induced depression has not yet been elucidated.

We have recently shown that neuropathic pain-induced depressive like behaviors in mice are associated with the upregulation of the mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the anterior cingulate cortex (ACC) [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF], a brain structure implicated in both pain-and mood-related processing [START_REF] Barthas | The anterior cingulate cortex is a critical hub for pain-induced depression[END_REF]. MKP-1 dephosphorylates both threonine and tyrosine residues of MAPKs thereby acting as an important negative regulator of the extracellular signal-regulated kinase (ERK) signaling cascades [START_REF] Jeffrey | Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses[END_REF]. The relation between MKP-1 and ERK is bidirectional as ERK signaling can also regulate transcription factors such as the cyclic-adenosine monophosphate (cAMP) response element binding protein (CREB) and transcription factor 1 (ATF1) found on the Mkp-1 promoter region [START_REF] Rastogi | Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways[END_REF]. The present study aimed to determine if neuropathic pain-induced depressive-like behaviors are associated with a disruption of this feedback loop and whether ketamine exerts its activity by targeting different members of the MAPK signaling pathway.

Specifically, the present study evaluated the effect of systemic ketamine administration on the mechanical hypersensitivity and depressive-like consequences of neuropathic pain as well as its effect on the MAPK signaling pathway. Our main results

showed that a single intraperitoneal (i.p.) injection of ketamine (15 mg/kg) results in rapid reduction of depressive-like behaviors lasting several days, while only transiently alleviating mechanical hypersensitivity. This ketamine-mediated phenotype was accompanied by a decrease in MKP-1, starting from 30 minutes (min) and lasting at least until 72 hours (h), and by an increase in phosphorylated ERK (pERK) in the ACC of neuropathic animals. The current findings shed light on the molecular alterations accompanying ketamine administration in the comorbidity of chronic pain and depression.

Materials and Methods

Animals

110 adult male C57BL/6J mice (Charles River, L'Arbresle, France) were used. The mice were 8 weeks old at the beginning of the experiments, housed 4 per cage and kept under a 12h light/dark cycle (lights on: 7 p.m and off: 7 a.m) with food/water availability ad libitum.

Results were obtained from a total of four independent cohorts of animals, wherein two were used for behavioral testing and two were used for western blot analyses. Protocols were approved by the University of Strasbourg ethics committee and performed according to animal care and use guidelines of the European Community Council Directive (EU 2010/63).

Neuropathic pain model and nociception assessment

Animals were anaesthetized with a combination of zoletil (25 mg/kg tiletamine and 25 mg/kg zolazepam) and xylazine (10mg/kg) (Centravet, Taden, France) i.p. before neuropathy was induced by unilateral implantation of a 2mm polyethylene tube (cuff) around the main branch of the right sciatic nerve (Yalcin et al., 2014b). Control (sham) mice received the same surgery without placing the cuff. The mechanical threshold was assessed before surgery (baseline) and on a weekly basis in the postoperative period with the von Frey test. During each session, the animals were individually habituated (10 min) in transparent, bottomless plastic boxes which were placed on a mesh platform. Next, filaments of different pressure (0.4 -8.0 g; Bioseb, Chaville, France) were applied to the ventral surface of each hindpaw in an ascending fashion. A positive response for a given pressure was recorded when 3 out of 5 applications resulted in withdrawal or licking of the stimulated hindpaw. The mechanical sensitivity threshold was characterized as a response to 2 consecutive filaments.

Pharmacological agents

Ketamine hydrochloride (Yliopiston Apteekki, Helsinki, Finland) was dissolved in 0.9% sodium chloride (NaCl) to a 3 mg/ml concentration and injected 0.10 -0.15 ml i.p., depending on the weight of the animal, to achieve a 15 mg/kg of ketamine dose per animal. Control animals received single i.p. injections of 0.9% NaCl.

Behavioral tests

The presence of anxiodepressive-like behaviors was assessed during the 8 week of post neuropathy induction. All the tests were performed during the animals' active phase (dark cycle), under red light and as previously described [START_REF] Yalcin | A time-dependent history of mood disorders in a murine model of neuropathic pain[END_REF]. The experimental design is detailed in Fig. 1A and Fig. 2A.

Novelty-suppressed feeding test

Food deprived (24 h) mice were placed in the corner of an open plastic box (40 cm x 40 cm x 30 cm) containing 2 cm of sawdust and a food pellet in the center. The latency to first contact and start eating the pellet was recorded within a time frame of 5 min after being placed in the box. The test measures the animal's motivation to approach the open space of the center of the arena where the food is located.

Splash test

This test involved spraying a 20% sucrose solution onto the dorsal coat and recording of animal´s total grooming activity over the next 5 min. A reduced grooming rate indicates a loss of motivation for self-hygiene, parallel to human apathy.

Forced swimming test

Each animal was slowly lowered into a glass cylinder (17.5 cm height x 12.5 cm diameter) with 12 cm of water (24°C). Two minutes after, the immobility time, which involved floating on the surface without active swimming movements, was recorded over the next 4 minutes.

Due to the stressful circumstances, this test was always performed last. The test measures the animal's helplessness-like behavior.

Tissue collection and protein analysis

For the molecular analyses, animals were sacrificed 30 min or 72 h after ketamine injection by cervical dislocation and the ACC was dissected and stored at -80°C. Next, protein extraction, followed by Western blot was performed. Protein extraction was started by mechanical dissociation of the tissues in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15% EDTA, 10% glycerol, 1% NP40) and centrifugation of the lysate (15,000 g/4°C/10 min).

Next, the supernatant was recuperated (100 μl) and a fraction of it used to determine its concentration with a protein assay (Quick Start Bradford, Bio-Rad, Munich, Germany) and spectrophotometry (Mitrhras LB940, Berthold Technologies). After adjusting the concentration of each sample to 1 μg/μl with lysis and Laemmli buffers, SDS-PAGE gel electrophoresis was done by separating 15 μl of the denatured proteins on 8% polyacrylamide gels and electroblotting them onto a polyvinylidene fluoride (PVDF) membrane (Millipore).

Finally, the membrane was incubated over night at 4°C in the primary antibody (anti-MKP-1, ab195261 lot GR239206-8 rabbit monoclonal, 1:60000; Abcam or pERK Phospho-p44/42 MAPK (Erk1/2) ref 9101 lot 28 rabbbit monoclonal; 1:600 Cell signaling), washed with TBST, and then incubated in the secondary antibody for 1h under agitation (AP370P Milipore lot 2899737; goat anti-rabbit; 1:10000 or 1:7500, respectively). Imaging was performed with the enhanced chemiluminescence detection system (ECL Amersham) using the Amersham Imager 680 system. The relative protein expression was calculated with the densitometry tool of Adobe Photoshop CS3 software.

Statistical analysis

All graphical results are expressed as mean ± SEM (standard error of mean). Statistical analyses were performed with STATISTICA 7.1 (Statsoft, Tulsa, Oklahoma) by using multifactor analysis of variance (ANOVA), with independent (Two-way ANOVA) or repeated measures and Duncan post hoc analyses. The significance level was set at p ≤ 0.05.

For detailed information see Supplementary table S1.

Results

Single ketamine administration transiently relieves neuropathy-induced mechanical hypersensitivity

Prior to the sciatic nerve cuffing surgery, we evaluated the mechanical threshold for nociceptive sensitivity using the von Frey filaments and organized the groups in such a way that their baseline sensitivity would be equal (Fig. 1B andC). After neuropathy induction, both sham operated and neuropathic mice were divided into a group which later received ketamine and one which received saline, resulting in a total of 4 different groups. Before ketamine administration, cuff-implanted animals consistently showed mechanical hypersensitivity in the ipsilateral paw, which lasted more than eight weeks after the surgery (Fig. 1C; F (4, 100) = 6.86, p ≤ 0.001). At 8 weeks, we first established the time-response curve of single dose of ketamine (15 mg/kg, i.p.) on mechanical sensitivity. We observed that ketamine alleviated the decreased mechanical threshold observed in neuropathic animals 3 h after the administration but this effect was no longer present at 24 h post-treatment (Fig. 1D and D', p ≤ 0.001). This rapid but transitory allodynia relief was not observed in neither sham nor neuropathic animals administered with saline (Fig. 1D andD'). Since the motor performance is an important cofounder for behavioural tests, we assessed the locomotor activity of animals 8 weeks after the surgery. The results confirmed our previous observation that the cuff surgery does not affect spontaneous activity (Fig. 1E, [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF][START_REF] Barthas | The anterior cingulate cortex is a critical hub for pain-induced depression[END_REF][START_REF] Sellmeijer | Hyperactivity of Anterior Cingulate Cortex Areas 24a/24b Drives Chronic Pain-Induced Anxiodepressive-like Consequences[END_REF][START_REF] Yalcin | A time-dependent history of mood disorders in a murine model of neuropathic pain[END_REF]) and also demonstrated for the first time that ketamine does not alter the locomotor activity at 1h after its administration, the time point at which we performed our first behavioral test (Fig. 1E).

Single ketamine administration ameliorates depressive-like behaviors in neuropathic mice

Compared to the control animals which received a sham surgery, peripheral nerve injury resulted in depressive behaviors 8 weeks after the surgery, as displayed by an increased latency to feed in the NSF test (Fig. 2B; p ≤ 0.05), a decreased grooming duration in the Splash test (Fig. 2C; p ≤ 0.05) and an increased immobility in the FST (Fig. 2D; p ≤ 0.001).

While the anti-allodynic effect was transient, a single injection of a subanesthetic dose of ketamine was sufficient to reduce neuropathy-induced depressive-like behaviors for a prolonged period of time (Fig. 2B-E). Specifically, the neuropathic animals showed a decrease in depression-related behaviors 1h after ketamine administration in the NSF test (Fig. 2B; p ≤ 0.01), 24 h after in the Splash test (Fig. 2C; p ≤ 0.05) and 72 h after in the FST (Fig. 2D; p ≤ 0.05). However, the antidepressant-like effect of ketamine ceased 2 weeks after the treatment since we observed no differences between saline and ketamine treated cuff animals (Fig. 2E; p > 0.05).

Single ketamine administration restores the disrupted MAPK signaling pathway

The level of the MKP-1 in the ACC was evaluated 30 min and 72 h after the ketamine administration using two different batches. Western blot analysis showed that MKP-1 protein level was increased in the ACC of saline injected animals displaying neuropathic paininduced anxiodepressive-like behaviors both at 30 min (Fig. 3A;F (1, 19) = 4.08, p ≤ 0.05; post hoc: sham saline < NP saline, p ≤ 0.01) and 72 h (Fig. 3C;F (1, 18) = 8.69, p ≤ 0.01; post hoc: sham saline < NP saline, p ≤ 0.01). This increase was diminished 30 minutes and 72 hours after single ketamine administration (Fig. 3A; p ≤ 0.01; Fig. 3C; p ≤ 0.01). Similarly, neuropathic animals displayed a decreased p-ERK protein level in the ACC (Fig. 3B;F (1, 20) = 0.42, p ≤ 0.05; post hoc: sham saline > NP saline, p ≤ 0.01) which was restored 30 min after single ketamine administration (Fig. 3B; NP saline < NP ketamine, p ≤ 0.05).

Discussion

By using a mouse model of the comorbidity of neuropathic pain and depression, the present study demonstrated that systemic administration of a single sub-anesthetic dose of ketamine (15 mg/kg) is sufficient to: i) transiently alleviate mechanical hypersensitivity; ii) decrease depressive-like behaviors induced by chronic pain for up to 72 h; iii) restore the increased MKP-1 and the decreased p-ERK protein levels in the ACC.

Treating chronic pain and depression in an independent manner is challenging, but the comorbidity of these disorders is far more difficult [START_REF] Bair | Depression and pain comorbidity: a literature review[END_REF]. Subclasses of classical antidepressant drugs such as tricyclic antidepressants, serotonin noradrenaline reuptake inhibitors and anticonvulsants such as gabapentin and pregabaline [START_REF] Gilron | Neuropathic pain: principles of diagnosis and treatment[END_REF] remain among the first line treatments for many chronic pain conditions as they have direct analgesic effects. Additionally, other treatments such as topical lidocaine, cannabinoids and opioids are used [START_REF] Attal | EFNS guidelines on the pharmacological treatment of neuropathic pain: 2010 revision[END_REF][START_REF] Dworkin | Recommendations for the pharmacological management of neuropathic pain: an overview and literature update[END_REF]. Even with these possibilities, there is still a lack of efficiency, and a considerable amount of patients experiencing side effects [START_REF] Chou | The effectiveness and risks of long-term opioid therapy for chronic pain: a systematic review for a National Institutes of Health Pathways to Prevention Workshop[END_REF][START_REF] Finnerup | The evidence for pharmacological treatment of neuropathic pain[END_REF], which poses an urgent need for alternative pain remedies. In the current study, we demonstrated that a single systemic administration of ketamine (15 mg/kg) alleviated mechanical allodynia in nerve injured mice 3 h after the administration, but the hypersensitivity was restored already at 24 h post-injection. This transient anti-nociceptive effect of a single ketamine administration is in accordance with previous results [START_REF] Koizuka | Systemic ketamine inhibits hypersensitivity after surgery via descending inhibitory pathways in rats[END_REF][START_REF] Qian | Systemic ketamine attenuates nociceptive behaviors in a rat model of peripheral neuropathy[END_REF].

One of the main targets of ketamine are the NMDA receptors which are crucial in the development and maintenance of central sensitization [START_REF] Petrenko | The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review[END_REF], characterized by an increase in dorsal horn excitability, resulting in hypersensitivity, hyperalgesia and allodynia [START_REF] Woolf | Central sensitization: implications for the diagnosis and treatment of pain[END_REF]. Thus, by inhibiting NMDA receptors, ketamine has been shown effective in the alleviation of pain in patients suffering from various conditions, including post-surgical pain [START_REF] Stubhaug | Mapping of punctuate hyperalgesia around a surgical incision demonstrates that ketamine is a powerful suppressor of central sensitization to pain following surgery[END_REF], fibromyalgia [START_REF] Graven-Nielsen | Ketamine reduces muscle pain, temporal summation, and referred pain in fibromyalgia patients[END_REF], complex regional pain syndrome [START_REF] Schwartzman | Outpatient intravenous ketamine for the treatment of complex regional pain syndrome: a double-blind placebo controlled study[END_REF] and neuropathic pain [START_REF] Jorum | Cold allodynia and hyperalgesia in neuropathic pain: the effect of N-methyl-D-aspartate (NMDA) receptor antagonist ketamine--a doubleblind, cross-over comparison with alfentanil and placebo[END_REF]. However, it is necessary to utilize higher cumulative doses, as well as prolonged serial infusions to achieve a longer lasting effect following the treatment [START_REF] Niesters | Ketamine for chronic pain: risks and benefits[END_REF][START_REF] Sigtermans | Ketamine produces effective and long-term pain relief in patients with Complex Regional Pain Syndrome Type 1[END_REF]. Curiously, a recent study using the same neuropathy model as here showed that a prolonged ketamine treatment (twice a day for 10 days) with the same dose (15 mg/kg i.p.) had different results depending on the time period of the drug administration [START_REF] Salvat | A comparison of early and late treatments on allodynia and its chronification in experimental neuropathic pain[END_REF]. Indeed, when ketamine was administered before neuropathic pain surgery, progressive recovery of mechanical allodynia was observed until 2 months after the cessation of the treatment. However, starting ketamine administration with a 25-day delay after neuropathy induction produced only partially recovery during the 10-day treatment, and allodynia completely returned 2 weeks post-treatment. These results suggest that ketamine efficiency is not only dependent on the dose and frequency of administration, but also on the temporal progression of chronic pain (i.e. development vs. maintenance phase).

Besides its effect on the somatosensory component of chronic pain, here we showed that a single injection of ketamine at a sub-anesthetic dose is sufficient to relieve neuropathic pain-induced depressive-like behaviors for at least 72 h, well beyond the acute pharmacological effects (elimination t1/2 ~10-15 min). The current study is the first to use a mouse model of comorbid neuropathic pain and depressive-like behaviors to show the antidepressant-like effect of acute ketamine administration, which has previously been shown only in rats [START_REF] Wang | A single subanesthetic dose of ketamine relieves depression-like behaviors induced by neuropathic pain in rats[END_REF] and in some clinical case studies [START_REF] Bigman | Use of ketamine for acute suicidal ideation in a patient with chronic pain on prescribed cannabinoids[END_REF][START_REF] Weber | Case Report of Subanesthetic Intravenous Ketamine Infusion for the Treatment of Neuropathic Pain and Depression with Suicidal Features in a Pediatric Patient[END_REF]. These results are also in accordance with previous data from stress-related rodent models of depressive-like behaviors [START_REF] Autry | NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses[END_REF][START_REF] Li | mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists[END_REF]) and human patients with major depressive disorder [START_REF] Ballard | Improvement in suicidal ideation after ketamine infusion: relationship to reductions in depression and anxiety[END_REF][START_REF] Berman | Antidepressant effects of ketamine in depressed patients[END_REF][START_REF] Zarate | A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression[END_REF] showing that ketamine is a rapid-acting, long-lasting antidepressant agent whose therapeutic effects are manifested within hours and sustain for several days. This is in contrast to the generally prescribed, monoaminergic-related drugs which take several weeks or even months to manifest their benefits [START_REF] Insel | The STAR*D trial: revealing the need for better treatments[END_REF][START_REF] Machado-Vieira | The Timing of Antidepressant Effects: A Comparison of Diverse Pharmacological and Somatic Treatments[END_REF]. Thus, it is of great interest in the field of psychiatric disorders, notably depression, to understand the physiochemical mechanisms behind the fast-acting, long-term antidepressant properties of ketamine [START_REF] Abdallah | Ketamine as a promising prototype for a new generation of rapid-acting antidepressants[END_REF][START_REF] Kavalali | How does ketamine elicit a rapid antidepressant response?[END_REF].

We then studied where and how ketamine acts. The transient antinociceptive and prolonged antidepressant effect of acute ketamine administration observed in the current study highlights the distinct sensory and affective responses to chronic pain. The fact that the duration of ketamine's antidepressant benefits surpasses its anti-nociceptive action suggests that the analgesic properties of ketamine might be mediated at the spinal and peripheral level [START_REF] Koizuka | Systemic ketamine inhibits hypersensitivity after surgery via descending inhibitory pathways in rats[END_REF][START_REF] Oatway | Peripheral antihyperalgesic and analgesic actions of ketamine and amitriptyline in a model of mild thermal injury in the rat[END_REF][START_REF] Sawynok | Modulation of formalin-induced behaviors and edema by local and systemic administration of dextromethorphan, memantine and ketamine[END_REF], whereas its antidepressant effects require the recruitment of cortical and limbic areas such as the ACC, hippocampus and amygdala [START_REF] Li | The behavioral deficits and cognitive impairment are correlated with decreased IGF-II and ERK in depressed mice induced by chronic unpredictable stress[END_REF][START_REF] Moghaddam | Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex[END_REF][START_REF] Niesters | Effect of subanesthetic ketamine on intrinsic functional brain connectivity: a placebo-controlled functional magnetic resonance imaging study in healthy male volunteers[END_REF]. Among these brain structures, the ACC seems to be critical for chronic pain induced depression [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF][START_REF] Barthas | The anterior cingulate cortex is a critical hub for pain-induced depression[END_REF] as well as for the antidepressant action of ketamine. For instance, Perrine et al. [START_REF] Perrine | Ketamine reverses stress-induced depression-like behavior and increased GABA levels in the anterior cingulate: an 11.7 T 1H-MRS study in rats[END_REF] showed that ketamine administration resulted in an increase of GABA levels in the ACC of rats subjected to chronic unpredictable stress. This is in line with previous studies showing a decreased level of GABA in the ACC of depressed patients [START_REF] Bhagwagar | Low GABA concentrations in occipital cortex and anterior cingulate cortex in medicationfree, recovered depressed patients[END_REF], as well as an overall depressionassociated hyperactivity of the ACC in both rodents [START_REF] Sellmeijer | Hyperactivity of Anterior Cingulate Cortex Areas 24a/24b Drives Chronic Pain-Induced Anxiodepressive-like Consequences[END_REF] and humans [START_REF] Drevets | Functional anatomical correlates of antidepressant drug treatment assessed using PET measures of regional glucose metabolism[END_REF]. Interestingly, it has been recently shown that acute ketamine reduces hyperactivity of ACC neurons induced by chronic pain in rats [START_REF] Zhou | Ketamine reduces aversion in rodent pain models by suppressing hyperactivity of the anterior cingulate cortex[END_REF], which might point to a potential mechanism through which it also exerts its antidepressant activity.

Accordingly, we performed all the mechanistic studies in the ACC.

Since the activation of NMDA receptors is related to an increase in intracellular MAPKs, including ERK, p38 and the c-Jun N-terminal kinase (JNK) [START_REF] Crown | Increases in the activated forms of ERK 1/2, p38 MAPK, and CREB are correlated with the expression of at-level mechanical allodynia following spinal cord injury[END_REF]Ji et al., 2009;[START_REF] Waxman | N-methyl-D-aspartate receptor subtype mediated bidirectional control of p38 mitogen-activated protein kinase[END_REF] and our neuropathic pain induced depression model induces alterations in this pathway in the ACC as well, we decided to focus on the impact of ketamine on the MAPK pathway in the ACC. Our results showed that an acutely administered ketamine attenuates the disrupted MAPK signaling pathway in the ACC of mice displaying neuropathic pain-induced depressive-like behaviors. Specifically, ketamine lowered the elevated ACC MKP-1 protein level in neuropathic mice at 30 min and 72 h after the drug administration, while it had no effect on the expression of MKP-1 in the ACC of control mice.

In addition, ketamine restored the decreased p-ERK in the ACC of mice with comorbid neuropathic pain and depressive-like behaviors. These results are supported by recent evidences from pain field suggesting that ketamine's analgesic activity might be partly mediated through the modulation of several members of the MAPK pathways [START_REF] Choi | Low dose ketamine reduces the induction of ERK1/2 and CREB signaling protein in a neuropathic pain model of rats[END_REF][START_REF] Kwon | Ketamine reduces the induced spinal p38 MAPK and pro-inflammatory cytokines in a neuropathic rats[END_REF][START_REF] Mei | Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain[END_REF]. Specifically, it was shown that ketamine's analgesic effect is associated with an inhibition of both spinal astrocyte JNK activation [START_REF] Mei | Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain[END_REF] and the increased expression of p38 and phospho-p38 [START_REF] Kwon | Ketamine reduces the induced spinal p38 MAPK and pro-inflammatory cytokines in a neuropathic rats[END_REF] as well as with a decreased upregulated ERK [START_REF] Choi | Low dose ketamine reduces the induction of ERK1/2 and CREB signaling protein in a neuropathic pain model of rats[END_REF] in the spinal cord of rats with neuropathic pain. In accordance with these findings, it has been shown that a single dose of subanesthetic ketamine which recruits the MAPK signaling cascade (Kohtala et al., 2019;[START_REF] Li | mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists[END_REF]) also ameliorates chronic stress-induced deficits in spine number and function [START_REF] Li | Glutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits caused by chronic stress exposure[END_REF], a common characteristic in depression [START_REF] Banasr | Cell atrophy and loss in depression: reversal by antidepressant treatment[END_REF]. Moreover, Réus et al. [START_REF] Reus | MAPK signaling correlates with the antidepressant effects of ketamine[END_REF] found that acute blockade of MAPK signaling is sufficient to induce depressive-like behaviors and prevent the antidepressant response to ketamine. Therefore, ketamine might exert its effect by altering the sustained negative regulation of MAPKs through MKP-1, which is thought to contribute to the neuronal atrophy and volume loss in limbic brain areas associated with depression [START_REF] Sheline | Hippocampal atrophy in recurrent major depression[END_REF][START_REF] Stockmeier | Cellular changes in the postmortem hippocampus in major depression[END_REF].

This has already been suggested as a potential mechanism of other pharmacological agents which alter the MAPK pathway in pain and depression-associated brain regions [START_REF] Duman | Signaling pathways underlying the pathophysiology and treatment of depression: novel mechanisms for rapid-acting agents[END_REF]. For instance, evidences suggest that antidepressants such as fluoxetine and imipramine might act by restoring the dysregulated expression of MKP-1 and ERK in the hippocampus and ACC [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF][START_REF] Duric | A negative regulator of MAP kinase causes depressive behavior[END_REF][START_REF] Yasuda | Imipramine ameliorates painrelated negative emotion via induction of brain-derived neurotrophic factor[END_REF].

Besides the ACC, ketamine has been shown to alter the expression of Mkp-1 in brain regions like the striatum or hippocampus [START_REF] Ficek | Molecular profile of dissociative drug ketamine in relation to its rapid antidepressant action[END_REF]. The reason we focused solely on the ACC is the absence of MKP-1 alterations in other brain regions in our mouse model of chronic pain-induced depression, including the hippocampus and the somatosensory cortex [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF].

While our results suggest that upregulated MKP-1 contributes to increased dephosphorylation of ERK, which, in turn, fosters the development of depression, the relationship of MKP-1 and ERK does not seem to follow a linear direction, and this pattern of altered expression does not always seem to be the case. In fact, some previous studies show that both chronic stress and neuropathic pain are associated with an increase in ERK activation in the ACC [START_REF] Kuipers | Molecular correlates of impaired prefrontal plasticity in response to chronic stress[END_REF][START_REF] Wei | Activation of Erk in the anterior cingulate cortex during the induction and expression of chronic pain[END_REF], and that this activation contributes to the induction of affective pain, including aversion in response to painful stimuli [START_REF] Cao | Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction and expression of affective pain[END_REF][START_REF] Dai | Biphasic activation of extracellular signal-regulated kinase in anterior cingulate cortex distinctly regulates the development of pain-related anxiety and mechanical hypersensitivity in rats after incision[END_REF]. Additionally, by combining chronic constriction injury and chronic mild stress, Bravo et al. [START_REF] Bravo | Depressive-like states heighten the aversion to painful stimuli in a rat model of comorbid chronic pain and depression[END_REF] showed that rats with comorbid chronic pain and depressive-like behaviors show a robust increase of ERK in the ACC. Moreover, Yasuda et al. [START_REF] Yasuda | Imipramine ameliorates painrelated negative emotion via induction of brain-derived neurotrophic factor[END_REF] demonstrated that chronic constriction injury induces an up-regulation of pERK1/2 in the ACC of rats, while treatment with the tricyclic antidepressant imipramine successfully reduced this overexpression. The observed discrepancies might stem from the intricate bi-directional relationship between MKP-1 and ERK, which might be differently regulated depending on the condition and specific cells and networks. Namely, although MKP-1 is the negative regulator of ERK [START_REF] Boutros | Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer[END_REF][START_REF] Sun | MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo[END_REF], it has been demonstrated that ERK can induce mkp-1 gene expression at the transcriptional level [START_REF] Brondello | The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade[END_REF], as well as enhance its phosphatase activity [START_REF] Slack | Distinct binding determinants for ERK2/p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1[END_REF], reflecting its role in a negative feedback control. On the other hand, it is also known that activated ERK can trigger MKP-1 proteolysis via the ubiquitin-proteasome pathway, hence achieving a positive feedback loop by decreasing its phosphatase activity [START_REF] Lin | ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway[END_REF]. These findings suggest that, depending on whether kinase activity needs to be sustained or inhibited, ERK has a dual function in regulating MKP-1 stability, which is achieved through docking to its different domains [START_REF] Lin | Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling[END_REF].

In addition to NMDA receptor antagonism and MAPK inhibition, ketamine produces indirect opioid system activation, demonstrated first in clinical studies [START_REF] Williams | Attenuation of antidepressant and antisuicidal effects of ketamine by opioid receptor antagonism[END_REF][START_REF] Williams | Attenuation of Antidepressant Effects of Ketamine by Opioid Receptor Antagonism[END_REF]. Recently Malinow's group showed that ketamine does not act as an opiate, but its effects require both NMDA and opiate receptor signaling, suggesting that interactions between these two neurotransmitter systems are necessary to achieve an antidepressant effect [START_REF] Klein | Cortical Excitability and Activation of TrkB Signaling During Rebound Slow Oscillations Are Critical for Rapid Antidepressant Responses[END_REF]. Besides its antidepressant effect, the implication of opioid receptors has also been shown in the analgesic effect of ketamine. Indeed, the antagonists of μ-and δ-but not kappa-opioid receptors block ketamine-induced central antinociception [START_REF] Pacheco Dda | Central antinociception induced by ketamine is mediated by endogenous opioids and mu-and delta-opioid receptors[END_REF].

In conclusion, this study clearly showed that ketamine could serve as alternative treatment for neuropathic pain patients with major depressive disorder as it has a dual effect on both the somatosensory and emotional consequences of chronic pain. While our study shows the sustained effect of ketamine on MAPK pathway, the present study could not directly test the causal link between ketamine-induced changes in MKP-1 and the relief of depressive-like symptoms. Indeed, since the pharmacological blockade or genetic suppression of MPK-1 being sufficient per se to suppress or prevent depressive-like behaviors in animal models [START_REF] Barthas | Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression[END_REF][START_REF] Duric | A negative regulator of MAP kinase causes depressive behavior[END_REF], the direct testing of the role of MKP-1 downregulation in ketamine action becomes very difficult.

Funding information

Figure legends

  This work was supported by the Centre National de la Recherche Scientifique (contract UPR3212), the University of Strasbourg, NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation (24736), Fondation pour la Recherche Médicale (FRM FDT201805005527), French National Research Agency (ANR) through the Programme d'Investissement d'Avenir under the contract ANR-17-EURE-0022. This study was also supported by The Scientific and Technological Research Council of Turkey (TUBITAK) through the 2219 international post-doctoral research fellowship program and the Academy of Finland (grant nro. 276333). We would like to thank Chronobiotron for animal care. Declaration of Competing Interest: None. The authors declare that they have no conflict of interest. The funding sources had no role in the study design, in the interpretation of data and in the writing of the manuscript. Ethical Statement Protocols were approved by the University of Strasbourg ethics committee and performed according to animal care and use guidelines of the European Community Council Directive (EU 2010/63).

Fig. 1 .

 1 Fig. 1. Antinociceptive effects of single systemic ketamine administration. A) Timeline of surgical and behavioral procedures. B) Pre-ketamine treatment mechanical sensitivity showing no difference in the post-surgery threshold of the contralateral paw of sham and neuropathic (NP) mice as assessed by von Frey test. C) von Frey test results from different time points during the post-surgery period showing a decreased mechanical sensitivity threshold of the ipsilateral paw of NP mice compared to sham-operated controls. D) von Frey test results after single ketamine, showing an increase in the mechanical sensitivity in the ipsilateral paw of NP mice at 3 h (D'), but not 24 h, after administration. E) Neither the peripheral nerve injury nor ketamine treatment affects the spontaneous locomotor activity.

Fig. 2 .

 2 Fig. 2. Antidepressant-like effect of single systemic ketamine administration.A) Timeline of surgical and behavioral procedures. B) NSF test showing a decrease in the latency to feed of NP mice 1 h after ketamine injection. C) Acute ketamine treatment resulted in an increased grooming duration of NP animals in the splash test 24 h later. D) Ketaminetreated NP mice showed a decrease in immobility time during FST 72 h after administration. E) Two weeks after ketamine injection, there was no difference in the grooming duration in the splash test between treated and non-treated NP mice, suggesting that the antidepressantlike effect of acute ketamine was no longer present. Sample sizes are presented in brackets next to experimental groups.

Fig. 3 .

 3 Fig. 3. The effect of ketamine on the MAPK pathway in the ACC. A) Western blot analysis showing an increase in MKP-1 protein level in the ACC of vehicle treated NP mice, and a decrease at 30 min after acute systemic ketamine treatment. B) Western blot results