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International audience; Background: Early life stress is known to affect the development of the nervous system and its function at a later age. It increases the risk to develop psychiatric disorders as well as chronic pain and its associated affective comorbidities across the lifespan. GABAergic inhibition is important for the regulation of central function and related behaviors, including nociception, anxiety or social interactions, and requires low intracellular chloride levels. Of particular interest, the oxytocinergic (OTergic) system exerts potent anxiolytic, analgesic and pro-social properties and is known to be involved in the regulation of chloride homeostasis and to be impaired following early life stress.Methods: We used behavioral measures to evaluate anxiety, social interactions and pain responses in a rat model of neonatal maternal separation (NMS). Using quantitative PCR, we investigated whether NMS was associated with alterations in the expression of chloride transporters in the cerebrum and spinal cord. Finally, we evaluated the contribution of OTergic signaling and neuro-inflammatory processes in the observed phenotype.Results: NMS animals displayed a long-lasting upregulation of chloride importer Na-K-Cl cotransporter type 1 (NKCC1) expression in the cerebrum and spinal cord. Neonatal administration of the NKCC1 inhibitor bumetanide or oxytocin successfully normalized the anxiety-like symptoms and the lack of social preference observed in NMS animals. Phenotypic alterations were associated with a pro-inflammatory state which could contribute to NKCC1 upregulation.Conclusions: This work suggests that an impaired chloride homeostasis, linked to oxytocin signaling dysfunction and to neuro-inflammatory processes, could contribute to the sensori-affective phenotype following NMS.

Noise and high light illumination in the neonatal intensive care unit (NICU) are recognized as stressors that could alter the well-being and development of vulnerable preterm infants. This prospective observational study evaluated the pain behaviours of very preterm infants (VPIs) to sound peaks (SPs) and light levels variations (LLVs) in the NICU. We measured spontaneously occurring SPs and LLVs in the incubators of 26 VPIs over 10 h. Their behavioural responses were analysed through video recordings using the “Douleur Aigue du Nouveau-né” (DAN) scale. We compared the maximum DAN scores before and after environmental stimuli and the percentage of VPIs with a score ≥ 3 according to the type of stimuli. A total of 591 SPs and 278 LLVs were analysed. SPs of 5 to 15 dBA and LLVs significantly increased the maximum DAN scores compared to baseline. The occurrence of DAN scores ≥ 3 increased with both stressors, with a total of 16% of SPs and 8% of LLVs leading to quantifiable pain behaviour. Altogether, this study shows that VPIs are sensitive to SPs and LLVs, with a slighter higher sensitivity to SPs. The mechanisms leading to pain behaviours induced by noise and light changes should be evaluated further in the context of VPIs brain development. Our results provide further arguments to optimize the NICU sensory environment of neonatal units and to adapt it to the expectations and sensory abilities of VPIs.

Inflammatory processes are critical promoting factors of chronic pain states, mostly by inducing peripheral and central sensitization of the nociceptive system. These processes are associated with a massive increase in glutamatergic transmission, sometimes facilitated by spinal disinhibition. In this study, we used etifoxine, a non-benzodiazepine anxiolytic known to amplify inhibition mediated by gamma-aminobutyric acid type A (GABAA) receptors in pain processing regions, either directly (through allosteric modulation) or indirectly (through the synthesis of endogenous neurosteroids). We used different models of local inflammation to evaluate the possible direct action of etifoxine on analgesia and edema. Pain symptom and edema measurements were performed after intraplantar carrageenan injection or after topical ear inflammation. We found that etifoxine treatment was associated with reduced plantar surface temperature 24 h after intraplantar carrageenan injection. In this model, etifoxine also alleviated thermal hot and mechanical hyperalgesia. A similar finding was observed while analyzing pain symptoms in the late phase of the formalin test. In a model of ear inflammation, etifoxine appeared to have a moderate anti-edemic effect after topical application. This slight action of etifoxine on the limitation of inflammatory processes could be mediated in part by cyclo-oxygenase 1 activity inhibition. Etifoxine appears as a promising therapeutic tool contributing to the limitation of inflammatory pain symptoms. Since etifoxine is already prescribed as an anxiolytic in several countries, it could be a good candidate for the prevention of inflammatory-driven edema and hyperalgesia, although the precise mechanism of action relative to its anti-inflammatory potential remains to be elucidated.

More than 450 million people worldwide suffer from diabetes, or 1 in 11 people. Chronic hyperglycemia degrades patients’ quality of life and the development of neuropathic pain contributes to the burden of this disease. In this study, we used the mouse model of streptozocin-induced diabetic type 1 neuropathy to assess the analgesic potential of etifoxine. Etifoxine is a prescribed anxiolytic that increases GABAAA receptor function through a direct positive allosteric modulation effect and, indirectly, by stimulating the production of endogenous GABAA receptor positive modulators such as allopregnanolone-type neurosteroids. We show that a curative or preventive treatment strongly and durably reduces mechanical hyperalgesia and anxiety in diabetic neuropathic mice. This analgesic and neuroprotective effect on painful symptoms and emotional comorbidities is promising and should now be clinically evaluated.

International audience; Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

For a long time, the capacity of the newborn infant to feel pain was denied. Today it is clear that the nociceptive system, even if still immature, is functional enough in the newborn infant to elicit pain responses. Unfortunately, pain is often present in the neonatal period, in particular in the case of premature infants which are subjected to a high number of painful procedures during care. These are accompanied by a variety of environmental stressors, which could impact the maturation of the nociceptive system. Therefore, the question of the long-term consequences of early life stress is a critical question. Early stressful experience, both painful and non-painful, can imprint the nociceptive system and induce long-term alteration in brain function and nociceptive behavior, often leading to an increase sensitivity and higher susceptibility to chronic pain. Different animal models have been developed to understand the mechanisms underlying the long-term effects of different early life stressful procedures, including pain and maternal separation. This review will focus on the clinical and preclinical data about early life stress and its consequence on the nociceptive system.

More than 450 million people worldwide suffer from diabetes, or 1 in 11 people. Chronic hyperglycemia degrades patients’ quality of life and the development of neuropathic pain contributes to the burden of this disease. In this study, we used the mouse model of streptozocin-induced diabetic type 1 neuropathy to assess the analgesic potential of etifoxine. Etifoxine is a prescribed anxiolytic that increases GABAAA receptor function through a direct positive allosteric modulation effect and, indirectly, by stimulating the production of endogenous GABAA receptor positive modulators such as allopregnanolone-type neurosteroids. We show that a post-symptomatic or preventive treatment strongly and durably reduces mechanical hyperalgesia and anxiety in diabetic neuropathic mice. This analgesic and neuroprotective effect on painful symptoms and emotional comorbidities is promising and should now be clinically evaluated.

International audience; Neuropathic pain is frequently associated with anxiety and major depressive disorders, which considerably impact the overall patient experience. Favoring GABAergic inhibition through the pain matrix has emerged as a promising strategy to restore proper processing of nociceptive and affective information in neuropathic pain states. In this context, the non-benzodiazepine anxiolytic etifoxine (EFX), known to amplify GABAergic inhibition through positive modulation of GABAA receptors and neurosteroidogenesis, presents several advantages. Therefore, we sought to investigate the preclinical therapeutic potential of EFX on the somatosensory and affective components of neuropathic pain. Here, we used a murine model in which neuropathic pain was induced by the implantation of a compressive cuff around the sciatic nerve (mononeuropathy). We showed that the intraperitoneal EFX treatment for five consecutive days (50 mg/kg) relieved mechanical allodynia in a sustained manner. Besides its effect on evoked mechanical hypersensitivity, EFX also alleviated aversiveness of ongoing pain as well as anxiodepressive-like consequences of neuropathic pain following cuff-induced mononeuropathy. This effect was also seen 12 weeks after induction of the neuropathy when allodynia was no longer present. Analgesic and neuroprotective actions of EFX were also seen by the absence of neuropathic pain symptoms if a second sciatic nerve constriction injury was applied to the contralateral hindpaw. Mass spectrometry analysis revealed a normalization of brainstem serotonin levels in EFX-treated animals and an increase in norepinephrine. This study suggests that EFX presents promising therapeutic potential for the relief of both somatosensory and affective consequences of neuropathic pain, a beneficial effect that is likely to involve monoamine descending controls.

SUMMARYOxytocin orchestrates social and emotional behaviors through modulation of neural circuits in brain structures such as the central amygdala (CeA). The long-standing dogma is that oxytocin signaling in the central nervous system occurs exclusively via direct actions on neurons. However, several findings over the last decades showed that astrocytes actively participate in the modulation of neuronal circuits. Here, we investigate the degree of astrocytes’ involvement in oxytocin functions. Using astrocyte’ specific gain and loss of function approaches, we demonstrate that CeA astrocytes not only directly respond to oxytocin, but are actually necessary for its effects on neuronal circuits and ultimately behavior. Our work identifies astrocytes as a crucial cellular substrate underlying the promotion of a positive emotional state by oxytocin. These results further corroborate that astrocytes are key regulators of neuronal circuits activity by responding to specific neuropeptidergic inputs, and opens up new perspectives to understand how neuromodulators gate brain functions.

Chronic administration of medication can have an important impact on metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment but morphine metabolic processes in the central nervous system are poorly characterised. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major pending question. Thus, our aim was to characterise the in vivo metabolism and BBB permeability of morphine after long-term treatment at both central and peripheral levels. Mice were injected with morphine or saline solution for 8 consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three H; 15%, w/w). Mice were then euthanized and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled counterparts. We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. This suggests that morphine analgesic tolerance is not linked to an increase of morphine glucuronidation into M3G or an alteration of the drug's global BBB permeability. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study.

Background although neonatal pain management has seen huge improvements in the past years, many gaps between knowledge and practice still exist. Objective to give the reader the state of the art of actual pain management and treatment. Methods a literature review was done on the physiopathology of pain, sex differences in the perception of pain, epidemiology, non-pharmacological treatment and developmental care approach, pharmacological treatment with pharmacokinetics and pharmacodynamics approaches. Conclusion despite an increasing knowledge in the field of neonatal pain, many gaps and questions remain, especially relative to the lack of assessment, prevention and treatment of painful procedures, appropriate drugs and dosing to support the well-being and the brain development of this highly vulnerable population.

International audience; The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D3 (cholecalciferol) on pain symptoms. We found that cholecalciferol supplementation improved mechanical nociceptive thresholds in monoarthritic animals and reduced mechanical hyperalgesia and cold allodynia in a model of mononeuropathy. Transcriptomic analysis of cerebrum, dorsal root ganglia and spinal cord tissues indicate that cholecalciferol supplementation induces a massive gene dysregulation which, in the cerebrum, is associated with opioid signaling (23 genes), nociception (14), and allodynia (8), and, in the dorsal root ganglia, with axonal guidance (37 genes), and nociception (17). Among the identified cerebral dysregulated nociception-, allodynia-and opioid-associated genes, 21 can be associated with vitamin D metabolism. However, it appears that their expression is modulated by intermediate regulators such as diverse protein kinases and not, as expected, by the vitamin D receptor. Overall, several genes-Oxt, Pdyn, Penk, Pomc, Pth, Tac1, Tgfb1-encoding for peptides/hormones stand out as top candidates to explain the therapeutic benefit of vitamin D3 supplementation. Further studies are now warranted to detail the precise mechanisms of action but also the most favourable doses and time windows for pain relief.

Preterm infants (PTI) typically experience many painful and stressful procedures or events during their first weeks of life in a neonatal intensive care unit, and these can profoundly impact subsequent brain development and function. Several protective interventions during this sensitive period stimulate the oxytocin system, reduce pain and stress, and improve brain development. This review provides an overview of the environmental risk factors experienced by PTI during hospitalization, with a focus on the effects of pain, and early maternal separation. We also describe the long-term adverse effects of the simultaneous experiences of pain and maternal separation, and the potential beneficial effects of maternal vocalizations, parental contact, and several related processes, which appear to be mediated by the oxytocin system.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

International audience; Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

Objectives: Anxiety and adjustment disorders are among the most prevalent mental health conditions. This review focuses on γ-aminobutyric acid receptor type A (GABAAR)-mediated anxiolysis, describi...

International audience; 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, 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 and OT antihyperalgesia 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. The involvement of epigenetic changes in these alterations was confirmed since neonatal treatment with the histone deacetylase inhibitor SAHA, not only normalized nociceptive sensitivities but also restored OT receptor-mediated stress-induced analgesia and the endogenous antihyperalgesia in inflamed NMS rats. There is growing evidence in the literature that early life stress might impair the nociceptive system ontogeny and function. This study suggests that these alterations might be restored while stimulating OT receptor signaling or histone deacetylase inhibitors, using molecules that are currently available or part of clinical trials for other pathologies.

International audience; 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 effer-ent 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 noci-ception, 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, Pierrick Poisbeau, Valery Grinevich and Alexandre Charlet contributed equally to this work. as well as propose some reasons for the application of exogenous OT for the treatment of certain types of pain in human patients.

Background Pathological pain states are often associated with neuronal hyperexcitability in the spinal cord. Reducing this excitability could theoretically be achieved by amplifying the existing spinal inhibitory control mediated by GABAA receptors (GABAARs). In this study, we used the non-benzodiazepine anxiolytic etifoxine (EFX) to characterize its interest as pain killer and spinal mechanisms of action. EFX potentiates GABAAR function but can also increase its function by stimulating the local synthesis of 3α-reduced neurosteroids (3αNS), the most potent endogenous modulators of this receptor. Methods The efficacy of EFX analgesia and the contribution of 3αNS were evaluated in a rat model of mononeuropathy. Spinal contribution of EFX was characterized through changes in pain symptoms after intrathecal injections, spinal content of EFX and 3αNS, and expression of FosB-related genes, a marker of long-term plasticity. Results We found that a 2-week treatment with EFX (>5 mg/kg, i.p.) fully suppressed neuropathic pain symptoms. This effect was fully mediated by 3αNS and probably by allopregnanolone, which was found at a high concentration in the spinal cord. In good agreement, the level of EFX analgesia after intrathecal injections confirmed that the spinal cord is a privileged target as well as the limited expression of FosB/ΔFosB gene products that are highly expressed in persistent pain states. Conclusions This preclinical study shows that stimulating the production of endogenous analgesics such as 3αNS represents an interesting strategy to reduce neuropathic pain symptoms. Since EFX is already prescribed as an anxiolytic in several countries, a translation to the human clinic needs to be rapidly evaluated.

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers. While endogenous morphine levels are detectable using enzyme-linked immunosorbant assay (ELISA), mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. This study describes the preparation of a specific anti-M6G rabbit polyclonal antibody and its validation. The specificity of this antibody was assessed against 30 morphine-related compounds. Then, a M6G-specific ELISA-assay was tested to quantify M6G in the plasma of healthy donors, morphine-treated, and critically ill patients. The antibody raised against M6G displays a strong affinity for M6G, codeine-6-glucuronide, and morphine-3-6-glucuronide, whereas only weak cross-reactivities were observed for the other compounds. Both M6G-ELISA and LC-MS/MS approaches revealed the absence of M6G in the plasma of healthy donors (controls, n = 8). In all positive donors treated with morphine-patch (n = 5), M6G was detected using both M6G-ELISA and LC-MS/MS analysis. Finally, in a study on critically ill patients with circulating endogenous morphine (n = 26), LC-MS/MS analysis revealed that 73% of the positive-patients (19 of 26), corresponding to high M6G-levels in M6G-ELISA, contained M6G. In conclusion, we show that endogenous M6G can be found at higher levels than morphine in the blood of morphine-naive patients. With respect to the interest of measuring endogenous M6G in pathologies, we provide evidences that our ELISA procedure represents a powerful tool as it can easily and specifically detect endogenous M6G levels.

Patients with schizophrenia have often been described as insensitive to nociceptive signals, but objective evidence is sparse. We address this question by combining subjective behavioral and objective neurochemical and neurophysiological measures. The present study involved 21 stabilized and mildly symptomatic patients with schizophrenia and 21 control subjects. We applied electrical stimulations below the pain threshold and assessed sensations of pain and unpleasantness with rating scales, and Somatosensory Evoked Potentials (SEPs/EEG). We also measured attention, two neurochemical stress indices (ACTH/cortisol), and subjective VEPs/EEG responses to visual emotional stimuli. Our results revealed that, subjectively, patients’ evaluations do not differ from controls. However, the amplitude of EEG evoked potentials was greater in patients than controls as early as 50 ms after electrical stimulations and beyond one second after visual processing of emotional pictures. Such responses could not be linked to the stress induced by the stimulations, since stress hormone levels were stable. Nor was there a difference between patients and controls in respect of attention performance and tactile sensitivity. Taken together, all indices measured in patients in our study were either heightened or equivalent relative to healthy volunteers.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Les neurosteroides sont synthetises par les cellules du systeme nerveux a partir de cholesterol ou de precurseurs steroidiens circulants. Comme les autres steroides, ils exercent de puissants effets activationnels sur les fonctions cerebrales dont une partie est mediee par l’action sur des recepteurs membranaires. Cette revue fait le point sur les effets membranaires rapides des neurosteroides et leurs consequences fonctionnelles sur l’expression douloureuse en experimentation animale. A cote de leurs effets therapeutiques potentiels en application exogene, les neurosteroides endogenes sont impliques dans la modulation des messages « douloureux » peripheriques dans la grande majorite des structures relais du systeme nociceptif en situations physiologique ou pathologique. La meilleure connaissance des mecanismes qui controlent la neurosteroidogenese endogene devrait permettre a l’avenir d’ouvrir de nouvelles pistes therapeutiques pour le traitement des douleurs neuropathiques et des douleurs inflammatoires.

International audience; Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non-genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3alpha neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3beta isomer was inefficient. More interestingly, 3alpha5beta neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3alpha5beta neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3alpha5beta neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

International audience; Because of its severity, chronicity, resistance to usual therapy and its consequences on quality of life, neuropathic pain represents a real clinical challenge. Fundamental research on this pathology uses metabolic, pharmacological or traumatic models in rodents that reproduce the characteristic human pain symptoms. In 1996, Mosconi and Kruger morphologically described a model of peripheral neuropathy in which a cuff of polyethylene tubing was placed around the sciatic nerve in rats. In the present study, we evaluated the behavioral consequences of this neuropathic pain model in C57Bl/6J mice which is the main genetic background used for studies in transgenic mice. A short cuff of polyethylene tubing was unilaterally placed around the main branch of the sciatic nerve. It induced an ipsilateral heat thermal hyperalgesia lasting around 3 weeks, and a sustained ipsilateral mechanical allodynia lasting at least 2 months. We showed that this neuropathic pain model is insensitive to ketoprofen, a non-steroidal anti-inflammatory drug. Morphine treatment acutely suppressed the mechanical allodynia, but tolerance to this effect rapidly developed. The analysis of video recordings revealed that most aspects of spontaneous behavior remained unaffected on the long term, excepted for a decrease in the time spent at social interaction for the neuropathic mice. Using the elevated plus-maze and the marble-burying test, we also showed that neuropathic mice develop an anxiety phenotype. Our data indicate that sciatic nerve cuffing in mice is a pertinent model for the study of nociceptive and emotional consequences of sustained neuropathic pain.

The activity of GABAergic inhibitory interneurones located in lamina II of the spinal cord is of fundamental importance for the processing of peripheral nociceptive messages. We have recently shown that 3alpha-hydroxy ring A-reduced pregnane neurosteroids [3alpha5alpha-neurosteroids (3alpha5alphaNS)], potent allosteric modulators of GABA(A) receptors (GABA(A)Rs), are synthesized in the spinal cord and limit thermal hyperalgesia during inflammatory pain. Because changes in the expression of calcium-dependent protein kinases [protein kinase C (PKC)] are observed during pathological pain in the spinal cord, we examined the possible interactions between PKC and 3alpha5alphaNS at synaptic GABA(A)Rs. Using patch-clamp recordings of lamina II interneurones in the spinal cord of 15-20-day-old rats, we showed that synaptic inhibition mediated by GABA(A)Rs and its modulation by 3alpha5alphaNS in lamina II of the spinal cord largely depend on activation of PKC. Our experimental results suggested that activation of PKC locks synaptic GABA(A)Rs in a functional state precluding further positive allosteric modulation by endogenous and exogenous 3alpha5alphaNS. This effect was fully prevented by coadministration of chelerythrin, an inhibitor of PKC. Furthermore, application of chelerythrin alone rendered synaptic GABA(A)Rs hypersensitive to endogenously produced or exogenously applied 3alpha5alphaNS. These findings confirmed that there was a significant production of endogenous 3alpha5alphaNS in lamina II of the spinal cord but also indicated that PKC-dependent phosphorylation processes were tonically activated to control GABA(A)R-mediated inhibition under resting conditions. We therefore can conclude that PKC activation sets an upper limit to the functional plasticity of GABAergic transmission induced by endogenous neurosteroids.

In the lithium-pilocarpine model (Li-pilocarpine) of temporal lobe epilepsy, GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A) IPSCs) were recorded in dentate gyrus granule cells (GCs) from adult rat hippocampal slices. The properties of GABA(A) IPSCs were compared before and after superfusion of modulators in control conditions (Li-saline rats) and in Li-pilocarpine rats 24-48 h and 3-5 months (epileptic rats) after status epilepticus (SE). The mean peak amplitude of GABA(A) IPSCs increased by about 40% over Li-saline values in GCs 24-48 h after SE and remained higher in epileptic rats. In Li-pilocarpine rats, studied at 24-48 h after SE, diazepam (1 microm) lost 65% of its effectiveness at increasing the half-decay time (T(50%)) of GABA(A) miniature IPSCs (mIPSCs). Diazepam had no effects on mIPSC T(50%) in epileptic rats. The benzodiazepine ligand flumazenil (1 microm), acting as an antagonist in Li-saline rats, exhibited a potent inverse agonistic effect on GABA(A) mIPSCs of GCs from Li-pilocarpine rats 24-48 h and 3-5 months after SE. The neurosteroid allopregnanolone (100 nm), which considerably prolonged GABA(A) mIPSCs in Li-saline rats, totally lost its effect in rats studied 24-48 h after SE. However, this decrease in effectiveness was transient and was totally restored in epileptic rats. In addition to the up-regulation in the number of receptors at individual GC synapses, we propose that these 'epileptic' GABA(A) receptors possess benzodiazepine binding sites with altered allosteric properties. The failure of benzodiazepine and neurosteroid to potentiate inhibition early after SE may be a critical factor in the development of epileptogenesis and occurrence of seizures.

In addition to its presence in blood samples from morphine-treated patients and heroin addicts, morphine is endogenously synthesized from dopamine in mammals [1, 2]. However, the physiological relevance of endogenous morphine (eM) remains an open question. Several studies report an increase in eM in blood and urine samples in pathological conditions such as infection, inflammation, bulimia, anorexia and Parkinson's disease [2, 3]. For example, eM concentrations reached on average 2.5 ng ml−1 in the serum of patients suffering from systemic infections (sepsis), whereas healthy donors and control patients displayed no or very low eM concentrations [4]. Therefore, quantification of endogenous and exogenous morphine is of great interest for both clinical and forensic analyses. As both endogenous and exogenous morphine are present at low concentrations, the quantification method implemented must be very sensitive. Besides, no consensus yet exists regarding blood sample preparation for morphine measurement using ELISA. As lithium-heparinate (plasma) and serum samples are both routinely used in clinical practice, we compared the morphine concentrations using these two sampling methods. This study was approved by our institutional review board for human experimentation (CPP no. 08/62-protocol no. IVD1 EUDRACT no. 2008-A01345-50). After informed consent, samples were collected from patients requiring clinical laboratory analyses. Blood from 80 patients was collected in two types of sampling tubes, as follows: (i) for serum analysis, blood was collected in dry tubes (BD, Plymouth, UK; SSTII 377615), and serum was obtained after centrifugation (4°C, 1500g, 10 min); and (ii) for plasma analysis, blood was collected in lithium-heparinate tubes (BD; LH-PSTII 367378, 17 i.u. ml−1) and plasma was recovered after centrifugation (4°C, 1500g, 10 min). Collected serum and plasma were immediately stored at −80°C (for 3 weeks to 3 months). Samples were thawed only once, 15 min prior to ELISA testing. In these conditions, no effect of freezing on morphine concentrations has been noticed. Both plasma and serum morphine concentrations were quantified using a morphine-specific ELISA kit (ref. 213-0480; Immunalysis, Pomona, CA, USA) [4]. No cross-reactivity has been observed for morphine metabolites (morphine-6-glucuronide, morphine-3-glucuronide and codeine) and 75 other compounds (related to eM biosynthesis pathway or not; see also kit's datasheet). The plasma or serum samples were tested in duplicates (40 µl) at 21°C. The coefficient of variation values were between 0% and 8%. All samples with a higher coefficient of variation value were retested in order to obtain a coefficient of variation below or equal to 8%. Morphine standards (0–25 ng ml−1) were diluted in phosphate-buffered saline containing 1% bovine serum albumin (PBS-BSA; pH 7.2; w/v) for serum samples and in PBS-BSA 1% containing lithium-heparinate (17 i.u. ml−1) for plasma samples. The detection limit for morphine was 0.1 ng ml−1. In a set of control experiments, known concentrations of morphine (0.28 and 11 ng ml−1) were diluted in PBS, PBS–lithium-heparinate (17 i.u. ml−1, pH 7.2), PBS–BSA (1%; w/v) or PBS–lithium-heparinate–BSA buffer. Using the ELISA kit, no statistical differences in morphine concentrations were observed (n = 8, Mann–Whitney U-test, P > 0.3) when 0.28 ng ml−1 of morphine was diluted in PBS (0.26 ± 0.03 ng ml−1), PBS–lithium-heparinate [0.26 ± 0.02 ng ml−1; 95% confidence interval (CI) of difference with PBS −0.03 to 0.03], PBS–BSA (0.27 ± 0.02 ng ml−1; 95% CI of difference with PBS −0.04 to 0.02) or PBS–lithium-heparinate–BSA (0.28 ± 0.04 ng ml−1; 95% CI of difference with PBS −0.04 to 0.0). Likewise, no statistical difference (n = 6, Mann–Whitney U-test, P > 0.2) was observed for 11 ng ml−1 of morphine diluted in PBS (11.06 ± 0.58 ng ml−1), PBS–lithium-heparinate (11.42 ± 0.36 ng ml−1; 95% CI of difference with PBS −0.63 to 0.10), PBS–BSA (11.01 ± 0.5 ng ml−1; 95% CI of difference with PBS −0.25 to 0.55) or PBS–lithium-heparinate–BSA (11.36 ± 0.55 ng ml−1; 95% CI of difference with PBS −0.94 to 0.52). This experiment clearly demonstrates that lithium-heparinate and BSA do not introduce a bias in the detection of morphine using this particular ELISA kit. However, when serum and lithium-heparinate tubes were spiked with a known concentration of morphine prior to fresh blood collection (healthy donors, n = 6; 10 ng ml−1), 86 ± 7% (95% CI 79.8–92.2%) of the initial morphine concentration was recovered in plasma samples, but only 42 ± 3% (95% CI 37.9–46.4%) in serum samples (Mann–Whitney U-test, P < 0.01). Finally, 80 patients distributed in the following three groups were tested: (i) healthy donors (n = 16); (ii) morphine-treated patients (morphine skin patch 12–48 h before blood collection, 20 mg day−1, n = 8); and (iii) critically ill patients (n = 56, naive for morphine), including patients with no infection and no inflammation (n = 18), systemic inflammatory response syndromes (n = 8), severe symstemic inflammatory response syndrome (n = 16), sepsis (n = 4), severe sepsis (n = 2) and septic shock (n = 8). In healthy donors, eM was undetectable in both serum and plasma samples. In morphine-treated patients (Figure 1), the mean morphine concentration in serum samples ranged from 3.2 to 31 ng ml−1 (median 8.1 ng ml−1; 95% CI 5.3–20.1 ng ml−1), whereas plasma values ranged from 25.7 to 114.8 ng ml−1 (median 54.8 ng ml−1; 95% CI 41.9–85.0 ng ml−1). On average, morphine concentrations in serum were 78 ± 15% (95% CI 68–88%) lower than in plasma (Wilcoxon signed-rank test, P < 0.05). In critically ill patients, eM concentrations ranged between 0 (not detectable) and 23.6 ng ml−1 in serum samples (median 1.1 ng ml−1; 95% CI 1.9–4.8 ng ml−1) and between 0.15 and 76.3 ng ml−1 (median 2.8 ng ml−1; 95% CI 6.7–15.5 ng ml−1) in plasma samples. Again, serum concentrations of eM were lower (from +13 to −98%; mean 65 ± 32%; 95% CI 57–73%) compared with plasma (Wilcoxon signed-rank test, P < 0.01). The presence of morphine was confirmed by liquid chromatography coupled to tandem mass spectrometry analysis performed on plasma and serum from eight patients not treated with morphine but positive for eM, whereas morphine was not detected in patients negative for eM in ELISA (n = 4) [4]. As expected, no morphine was detected in samples from four healthy donors. Figure 1 Concentrations of morphine in serum expressed as a function of the corresponding morphine concentrations in plasma in critically ill patients (n = 56, triangles) and in morphine-treated patients (n = 8, crosses). Inset shows a higher magnification of ... Our results revealed that morphine concentrations were systematically higher in plasma compared with serum for 61 of 64 patients and thus indicate that lithium-heparinate samples give a more accurate quantification of morphine blood concentrations. The cause of such a difference in morphine quantification between serum and plasma using this specific ELISA kit is currently under investigation by our group and might involve morphine binding to blood proteins [5] other than serum albumin. In line with this possibility, morphine detection in the blood has been shown to be highly dependent on urea concentrations (chaotropic agent), suggesting a link between morphine-binding proteins and morphine bioavailability [5]. Likewise, lithium is also a chaotropic/denaturing agent; therefore, it might explain why this ELISA quantification method for morphine is more efficient in the presence of lithium. The present results clearly demonstrate that to obtain accurate ELISA determinations of exogenous and endogenous morphine concentrations in the blood, the analysis should be carried out on lithium-heparinate plasma samples rather than serum samples.

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic–pituitary–adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3a5a-reduced steroids, which modulate GABAA receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3a5a-reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non-genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C-fiber-mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

Etifoxine (EFX) is a non-benzodiazepine anxiolytic which potentiate GABAA receptor (GABAAR) function directly or indirectly via the production of 3α-reduced neurosteroids. The later effect is now recognized to account for the long-term reduction of pain symptoms in various neuropathic and inflammatory pain models. In the present study, we characterized the acute antinociceptive properties of EFX during spinal pain processing in naive and monoarthritic rats using in vivo electrophysiology. The topical application of EFX on lumbar spinal cord segment, at concentrations higher than 30 μM, reduced the excitability of wide dynamic range neurons receiving non-nociceptive and nociceptive inputs. Windup discharge resulting from the repetitive stimulation of the peripheral receptive field, and recognized as a short-term plastic process seen in central nociceptive sensitization, was significantly inhibited by EFX at these concentrations. In good agreement, mechanical nociceptive thresholds were also significantly increased following an acute intrathecal injection of EFX. The acute modulatory properties of EFX on spinal pain processing were never seen in the simultaneous presence of bicuculline. This result further confirmed EFX antinociception to result from the potentiation of spinal GABAA receptor function.

1. We have used the whole-cell configuration of the patch-clamp technique to investigate the effects of neuroactive steroids on GABAA receptor-mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre- and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor-mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half-maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) reversibly potentiated IGABA in a dose-dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/- 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta-isomer of allopregnanolone, epipregnanolone (5 beta-pregnan-3 beta-ol-20-one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5-pregnen-3 alpha-ol-20-one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of -80 and -60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre- and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

International audience; Inflammatory and degenerative diseases of the joint are major causes of chronic pain. Long-lasting pain symptoms are thought to result from a central sensitization of nociceptive circuits. These processes include activation of microglia and spinal disinhibition. Using a monoarthritic rat model of pain, we tried to potentiate neural inhibition by using etifoxine (EFX), a nonbenzodiazepine anxiolytic that acts as an allosteric-positive modulator of gamma-aminobutyric acid type A (GABAA) receptor function. Interestingly, EFX also can bind to the mitochondrial translocator protein (TSPO) complex and stimulate the synthesis of 3α-reduced neurosteroids, the most potent positive allosteric modulator of GABAA receptor function. Here we show that a curative and a preventive treatment with 50mg/kg of EFX efficiently reduced neuropathic pain symptoms. In the spinal cord, EFX analgesia was accompanied by a reduction in microglial activation and in the levels of proinflammatory mediators. Using electrophysiological tools, we found that EFX treatment not only amplified spinal GABAergic inhibition, but also prevented prostaglandin E2-induced glycinergic disinhibition and restored a "normal" spinal pain processing. Because EFX is already distributed in several countries under the trade name of Stresam for its anxiolytic actions in humans, new clinical trials are now required to further extend its therapeutic indications as pain killer.

As a first step towards elucidating mechanisms involved in neuroendocrine synaptogenesis, we developed a model of co-culture based on hypothalamic-intermediate pituitary interactions. Dissociated hypothalamic neurons from fetal rats at embryonic day 15 were cultured in a defined medium together with melanotrope cells of the pituitary intermediate lobe from neonatal rats. In these co-cultures, establishment of synaptic contacts between GABAergic or dopaminergic neurons and an endocrine target cell, the melanotrope cell, was studied by morphofunctional approaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and AE-melanocyte-stimulating hormone, we demonstrated morphological contacts between GABAergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synapsin I, we showed a modification of synapsin I immunoreactivity from diVuse to punctate distribution correlated with the establishment of contacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrophysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic currents, which occurred either spontaneously and/or could be evoked chemically by 50 mM KCl or 100 µM kainate. The proportion of the melanotrope cells receiving functional synaptic inputs increased until 10 days in culture, a stage at which virtually all melanotrope cells in contact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approaches. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd.

Morphine, codeine, morphine-6-glucuronide, and morphine-3-glucuronide are synthesized de novo in mammalian cells and in the central nervous system. Knowledge on endogenous morphine-like compound distribution in the adult mouse brain has been recently improved, and new hypotheses have been suggested about the potential implications in brain physiology. Endogenous morphine-like compounds have been shown to be synthesized in the spinal cord, but their localization is unknown. Here we describe the distribution of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the adult mouse spinal cord using a well-validated antibody. By using different microscopy approaches, we found the presence of morphine, codeine, or morphine glucuronides in γ-aminobutyric acid (GABA)-ergic neurons and astrocytes of the spinal cord. Whereas GABAergic neurons containing endogenous morphine-like compounds were located primarily in the ventral horn, astrocytes that were labeled for morphine-like compounds were found throughout the gray matter and the white matter. Our study demonstrates the possibility that endogenous morphine-like compounds in the central nervous system have other functions beyond their analgesic functions. J. Comp. Neurol. 520:1547–1561, 2012. © 2011 Wiley Periodicals, Inc.

The properties of neuronal-type nicotinic acetylcholine receptors (nAChRs) present on the neuroendocrine cells of the porcine pars intermedia of the pituitary were studied in intact single cell using measurements of the free intracellular Ca2+ concentration ([Ca]i) with the calcium-sensitive dye fura 2. Local application of an extracellular solution containing 50 mM K+ or of the selective nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) depolarised the cells and induced an elevation in [Ca]i. The effect of DMPP on [Ca]i was dose dependent (EC50 = 6 microM), reversibly blocked by d-tubocurarine and strictly dependent on the concentration of extracellular Ca2+. The calcium channel blocker Cd2+ (100 microM) reversibly blocked 80% of the response induced by 50 mM K+, whereas it reduced the DMPP response by only 50%. In the absence of extracellular Na+, DMPP no longer depolarised the cells but still increased [Ca]i. The rise in [Ca]i under these conditions represented 41% of the control response, i.e. in the presence of external Na+. Thus activation of nAChRs induced an elevation in [Ca]i which was in part independent of cell depolarisation. This was confirmed by recording simultaneously, under whole-cell voltage-clamp, a rise in [Ca]i associated with the inward nicotinic current. During prolonged application of the agonist (50 s), the amplitude of the nicotinic current decayed rapidly to a very low plateau level reflecting nAChR desensitisation. However, photometric experiments performed on intact non-dialysed cells revealed the presence of a slowly decaying phase in [Ca]i throughout the application of DMPP. This suggests the persistence of a substantial Ca2+ influx during prolonged exposure to the agonist. Taken together, our results show that stimulation of nAChRs induces an influx of Ca2+ which elevates [Ca]i. This phenomenon is due to activation of voltage-dependent Ca2+ channels and to Ca2+ entry through the nAChR.

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions. J. Comp. Neurol. 519:2390–2416, 2011. © 2011 Wiley-Liss, Inc.

Evaluation of pain is a critical issue in human pathologies but also in animal experimentation. In human studies there is growing evidence that cardiovascular outputs such as heart rate variability (HRV) might be of interest to detect and measure pain expression. Indeed, systems controlling cardiovascular function are closely coupled to the perception of pain. To demonstrate the interest of HRV, we have combined radiotelemetry and remote-controlled nociceptive tests in rats submitted to various situations of acute and persistent inflammatory pain. We found the Poincare plot descriptor SD1 and pNN18 to represent robust indicators of pain, especially in the case of persistent inflammatory states. Further studies will be performed in order to understand by which mechanisms pain-related increases in HRV are produced and if these descriptors can be used for other persistent pain states.

Although cholinergic drugs are known to modulate nociception, the role of endogenous acetylcholine in nociceptive processing remains unclear. In the current study, we evaluated the role of cholinergic transmission through spinal β(2)-subunit-containing nicotinic acetylcholine receptors in the control of nociceptive thresholds. We show that mechanical and thermal nociceptive thresholds are significantly lowered in β(2)(∗)-knockout (KO) mice. Using nicotinic antagonists in these mice, we demonstrate that β(2)(∗)-nAChRs are responsible for tonic inhibitory control of mechanical thresholds at the spinal level. We further hypothesized that tonic β(2)(∗)-nAChR control of mechanical nociceptive thresholds might implicate GABAergic transmission since spinal nAChR stimulation can enhance inhibitory transmission. Indeed, the GABA(A) receptor antagonist bicuculline decreased the mechanical threshold in wild-type but not β(2)(∗)-KO mice, and the agonist muscimol restored basal mechanical threshold in β(2)(∗)-KO mice. Thus, β(2)(∗)-nAChRs appeared to be necessary for GABAergic control of nociceptive information. As a consequence of this defective inhibitory control, β(2)(∗)-KO mice were also hyperresponsive to capsaicin-induced C-fiber stimulation. Our results indicate that β(2)(∗)-nAChRs are implicated in the recruitment of inhibitory control of nociception, as shown by delayed recovery from capsaicin-induced allodynia, potentiated nociceptive response to inflammation and neuropathy, and by the loss of high-frequency transcutaneous electrical nerve stimulation (TENS)-induced analgesia in β(2)(∗)-KO mice. As high-frequency TENS induces analgesia through Aβ-fiber recruitment, these data suggest that β(2)(∗)-nAChRs may be critical for the gate control of nociceptive information by non-nociceptive sensory inputs. In conclusion, acetylcholine signaling through β(2)(∗)-nAChRs seems to be essential for setting nociceptive thresholds by controlling GABAergic inhibition in the spinal cord.

Background-: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. Results-: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. Conclusions-: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

Effective relief of acute and long-term postoperative pain is of utmost importance to patients undergoing surgery. Here, we worked on a controlled procedure of abdominal surgery in the rat inducing persistent postoperative pain symptoms for up to 10 days and tested the efficacy of perioperative care with the local anesthetic ropivacaine. Laparotomy was likewise used to implant radiotelemetric probes by which electrocardiogram, body temperature, and locomotor activity were recorded in freely moving animals. We showed that postoperative pain symptoms (mechanical allodynia) measured in periphery of the scar were associated over time with persistent tachycardia, elevated heart rate variability, and loss of mobility. Furthermore, a single subcutaneous infiltration of the local anesthetic ropivacaine in the periphery of the abdominal incision was sufficient to prevent the appearance of allodynia and the associated cardiac and motor signs of pain, monitored by radio- telemetry. These beneficial effects were observed when the infiltration was performed in the perio- perative period, but not later. This study on freely moving animals exhibiting long-lasting postoperative pain symptoms and altered autonomic/motor function illustrates well the importance of the timing of preemptive analgesia care with long-acting local anesthetics. Moreover, it empha- sizes the utility of monitoring heart rate variability to quantify spontaneous expression of long- lasting postoperative pain. Perspective: Speeding the recovery time after surgery using perioperative ropivacaine care is of significant clinical relevance because it might limit the risk of chronic pain and postoperative compli- cations. In humans, chronobiological analysis of heart rate variability could also help quantify spon- taneous pain expression with minimal emotional bias. a 2011 by the American Pain Society

In animal studies, thermal sensitivity is mostly evaluated on the basis of nociceptive reaction latencies in response to a given thermal aversive stimulus. However, these techniques may be inappropriate to differentiate allodynia from hyperalgesia or to provide information differentiating the activation of nociceptor subtypes. The recent development of dynamic hot and cold plates, allowing computer-controlled ramps of temperature, may be useful for such measures. In this study, we characterized their interest for studying thermal nociception in freely moving mice and rats. We showed that escape behavior (jumps) was the most appropriate parameter in C57Bl/6J mice, whereas nociceptive response was estimated by using the sum of paw lickings and withdrawals in Sprague-Dawley rats. We then demonstrated that this procedure allows the detection of both thermal allodynia and hyperalgesia after peripheral pain sensitization with capsaicin in mice and in rats. In a condition of carrageenan-induced paw inflammation, we observed the previously described thermal hyperalgesia, but we also revealed that rats exhibit a clear thermal allodynia to a cold or a hot stimulus. These results demonstrate the interest of the dynamic hot and cold plate to study thermal nociception, and more particularly to study both thermal allodynia and hyperalgesia within a single paradigm in awake and freely moving rodents.Despite its clinical relevance, thermal allodynia is rarely studied by researchers working on animal models. As shown after stimulation of capsaicin-sensitive fibers or during inflammatory pain, the dynamic hot and cold plate validated in the present study provides a useful tool to distinguish between thermal allodynia and thermal hyperalgesia in rodents.

The central processing of peripheral nociceptive messages is highly controlled by the activity of local inhibitory networks in the spinal cord and supraspinal centers. Recently, it has been shown that endogenous 3alpha-reduced neurosteroids (3alphaNS) exert a significant spinal antinociception by potentiating GABA(A) receptor function. Because endogenous 3alphaNS can be produced in many relay structures of the nociceptive system, we tested the potential analgesic efficacy of promoting the production of neurosteroids by using etifoxine (ETX, 50mg/kg i.p.). This prescribed non-benzodiazepine anxiolytic was shown previously to stimulate neurosteroidogenesis in its early step after binding to the mitochondrial translocator protein complex (TSPO). Using an animal model of generalized neuropathic pain resulting from a 2-week treatment with the antitumoral agent vincristine sulfate (VCR, 0.1mg/kg i.p.), we show that injections of ETX (50mg/kg i.p.) given every day reduced the VCR-induced mechanical and thermal pain symptoms but also prevented their appearance, if used in prophylaxia 1 week before VCR. Both the curative and preventive effects of ETX on pain symptoms were mediated by the production of 3alphaNS as demonstrated in animals treated with the enzymatic inhibitor provera (6-medroxyprogesterone acetate; 20mg/kg s.c.). Altogether, this study shows for the first time that promoting 3alphaNS could be a possible therapeutic strategy to treat neuropathic pain symptoms. Since ETX is already available as an anxiolytic, its use in humans, provided that its analgesic properties are confirmed, could be rapidly considered.

Background: Recent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception. Results: We show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages. Conclusion: Our results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.

Devices designed for mechanical pain threshold studies are often difficult to implement. The purpose of this study was to investigate a simple tool based on calibrated forceps to induce quantifiable mechanical stimulation in the rat on a linear scale. The most suitable protocol was tested by determining the effects of 3 repetitive measurements on both hind paws, respectively, during long-term (9 days), mid-term (1 day), and short-term (2 hours). Only threshold increase related to weight gain over long-term was observed, suggesting that moderate rat training can be used. The capacity of the device to reveal hyperalgesia was tested in a model of carrageenan-induced inflammation in the hind paw. The hyperalgesia was maximal 6 hours after carrageenan injection and progressively decreased. Similar, although more variable, responses were observed with von Frey filaments. Morphine-induced analgesia resulted in a dose-dependent increase of paw threshold. Tolerance to morphine administrated on a once daily schedule (10 mg/kg) during 5 days was revealed by a significant decrease in analgesia by day 3. Taken together, these results demonstrated accuracy of this device for easy, fast, and reproducible measure of mechanical pain threshold on rat limbs. Moreover, it allows the performance of rat testing with minimal constraint, which reduces data variability. Perspective The calibrated forceps is an easy to use device well-suited to rapidly test mechanical pain threshold with accuracy. It is well-designed for preclinical behavioral screening of noxious or analgesic properties of molecules.