Search

Background and PurposePain management is a major health burden. Pain results from the integration of the nociceptive message and neuronal communication relying on neurotransmitters such as glutamate, γ-aminobutyric acid (GABA), dopamine, noradrenaline and serotonin in brain regions including the periaqueductal gray (PAG), the nucleus accumbens (Nac), the caudate-putamen (Cpu) and the amygdala. Morphine remains the gold standard painkiller for severe painviathe activation of the mu opioid receptors. However, among side effects, morphine chronic treatment lead to antinociceptive tolerance. As antinociceptive tolerance might be linked to neurotransmission dysregulation, we have compared various neurotransmitter concentrations in acutevschronic morphine conditions in the amygdala, PAG, Cpu, and the Nac of male and female mice.Experimental approachSex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. The behavioural effects of acute and chronic morphine treatments, as well as sex differences in the levels of dopamine, serotonin, noradrenaline, glutamate and GABA in the amygdala, PAG, Cpu, and the Nac were determined by an absolute quantification LC-MS/MS approach using the isotopic dilution method.Key resultsThis study indicates, as previously reported, that female mice are less sensitive to morphine and develop morphine antinociceptive tolerance earlier than males (ED50 of 5.5±0.24 daysvs1.54±0.11 days, respectively). However, the rate at which the tolerance developed did not differ between both sex. We have found major differences in dopamine, serotonin, noradrenaline, glutamate and GABA levels between female and male mice in the amygdala, PAG, Cpu, and the Nac. Finally, no major effect of anti-nociceptive tolerance induced by chronic morphine was observed compared to acute administration of morphine.ConclusionNeurotransmitter differences are attributable mainly to sex differences in pain-related CNS regions. However, the impacts of morphine anti-nociceptive tolerance on dopamine, serotonin, noradrenaline, glutamate and GABA contents appeared to be limited.

International audience; Cocaine addiction is a complex pathology inducing long-term neuroplastic changes that, in turn, contribute to maladaptive behaviors. This behavioral dysregulation is associated with transcriptional reprogramming in brain reward circuitry, although the mechanisms underlying this modulation remain poorly understood. The endogenous cannabinoid system may play a role in this process in that cannabinoid mechanisms modulate drug reward and contribute to cocaine-induced neural adaptations. In this study, we investigated whether cocaine self-administration induces long-term adaptations, including transcriptional modifications and associated epigenetic processes. We first examined endocannabinoid gene expression in reward-related brain regions of the rat following self-administered (0.33 mg/kg intravenous, FR1, 10 days) cocaine injections. Interestingly, we found increased Cnr1 expression in several structures, including prefrontal cortex, nucleus accumbens, dorsal striatum, hippocampus, habenula, amygdala, lateral hypothalamus, ventral tegmental area, and rostromedial tegmental nucleus, with most pronounced effects in the hippocampus. Endocannabinoid levels, measured by mass spectrometry, were also altered in this structure. Chromatin immunoprecipitation followed by qPCR in the hippocampus revealed that two activating histone marks, H3K4Me3 and H3K27Ac, were enriched at specific endocannabinoid genes following cocaine intake. Targeting CB1 receptors using chromosome conformation capture, we highlighted spatial chromatin re-organization in the hippocampus, as well as in the nucleus accumbens, suggesting that destabilization of the chromatin may contribute to neuronal responses to cocaine. Overall, our results highlight a key role for the hippocampus in cocaine-induced plasticity and broaden the understanding of neuronal alterations associated with endocannabinoid signaling. The latter suggests that epigenetic modifications contribute to maladaptive behaviors associated with chronic drug use.

Objectives Increased availability of high-calorie palatable food in most countries has resulted in overconsumption of these foods, suggesting that excessive eating is driven by pleasure, rather than metabolic need. The behavior contributes to the rise in eating disorders, obesity, and associated pathologies like diabetes, cardiac disease, and cancers. The mesocorticolimbic dopamine and homeostatic circuits are interconnected and play a central role in palatable food intake. The endocannabinoid system is expressed in these circuits and represents a potent regulator of feeding, but the impact of an obesogenic diet on its expression is not fully known. Methods Food intake and body weight were recorded in male Wistar rats over a 6-week free-choice regimen of high fat and sugar; transcriptional regulations of the endocannabinoid system were examined post-mortem in brain reward regions (prefrontal cortex, nucleus accumbens, ventral tegmental area, and arcuate nucleus). K-means cluster analysis was used to classify animals based on individual sensitivity to obesity and palatable food intake. Endocannabinoid levels were quantified in the prefrontal cortex and nucleus accumbens. Gene expression in dopamine and homeostatic systems, including ghrelin and leptin receptors, and classical homeostatic peptides, were also investigated. Results The free-choice high-fat -and sugar diet induced hyperphagia and obesity in rats. Cluster analysis revealed that the propensity to develop obesity and excessive palatable food intake was differently associated with dopamine and endocannabinoid system gene expression in reward and homeostatic brain regions. CB2 receptor mRNA was increased in the nucleus accumbens of high sugar consumers, whereas CB1 receptor mRNA was decreased in obesity prone rats. Conclusions Transcriptional data are consistent with observations of altered dopamine function in rodents that have access to an obesogenic diet and point to cannabinoid receptors as GPCR targets involved in neuroplasticity mechanisms associated with maladaptive intake of palatable food. Supplementary Information The online version contains supplementary material available at 10.1007/s00394-021-02613-0.

Upon stress exposure a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine, serotonin), and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using Graph Theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g. prefrontal cortex, amygdala, periventricular hypothalamus), and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum, and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network.GRAPHICAL ABSTRACTGRAPHICAL ABSTRACT TEXTIn this study, using immunohistochemical staining of the immediate early gene c-fos and graph theory-based functional correlational analyses, we aimed at unravelling the possible engagement of the lateral habenula (LHb) within the stress response network during acute stress exposure (10-min restraint) in rats. We found that the medial part of the LHb (LHbM) was preferentially engaged, and that this engagement was concomitant to this of structures such as the medial prefrontal cortex (mPFC), the insular cortex (Ins), hypothalamic (PVH) and thalamic (PVT) paraventricular nuclei, the extended amygdala, comprising the Bed nucleus of the stria terminalis (BNST) and the entire amygdala (AMG), as well as the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal raphe nucleus (RD). This suggests upon stressful situations the LHbM serves as a relay of cortical, thalamic, hypothalamic and temporal information, further transmitted to midbrain monoaminergic systems to probably initiate coping strategies.

International audience; Background and Purpose-In rodents, morphine antinociception is influenced by sex. However, conflicting results have been reported regarding the interaction between sex and morphine antinociceptive tolerance. Morphine is metabolised in the liver and brain into morphine-3-glucuronide (M3G). Sex differences in morphine metabolism and differential metabolic adaptations during tolerance development might contribute to behavioural discrepancies. This article investigates the differences in peripheral and central morphine metabolism after acute and chronic morphine treatment in male and female mice. Experimental Approach-Sex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. After acute and chronic morphine treatment, morphine and M3G metabolic kinetics in the blood were evaluated using LC-MS/MS. In addition, they were quantified in several central nervous system (CNS) regions. Finally, the blood-brain barrier (BBB) permeability of M3G was assessed in male and female mice. Key Results-This study demonstrated that female mice showed weaker morphine antinociception and faster induction of tolerance than males. Additionally, female mice showed higher levels of M3G in the blood and several pain-related CNS regions than male mice, whereas lower levels of morphine were observed in these regions. M3G brain/blood ratios after injection of M3G indicated no sex differences in M3G BBB permeability, and these ratios were lower than those obtained after injection of morphine. Conclusion-These differences are attributable mainly to morphine central metabolism, which differed between males and females in pain-related CNS regions, consistent with weaker morphine antinociceptive effects in females. However, the role of morphine metabolism in antinociceptive tolerance seemed limited.

International audience; Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patient with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.

Binge eating, the defining feature of binge eating disorder (BED), is associated with a number of adverse health outcomes as well as a reduced quality of life. Animals, like humans, selectively binge on highly palatable food suggesting that the behaviour is driven by hedonic, rather than metabolic, signals. Given the links to both reward processing and food intake, this study examined the contribution of the endocannabinoid system (ECS) to binge-like eating in rats. Separate groups were given intermittent (12 h) or continuous (24 h) access to 10% sucrose and food over 28 days, with only the 12 h access group displaying excessive sucrose intake within a discrete period of time (i.e., binge eating). Importantly, this group also exhibited alterations in ECS transcripts and endocannabinoid levels in brain reward regions, including an increase in cannabinoid receptor 1 (CB1R) mRNA in the nucleus accumbens as well as changes in endocannabinoid levels in the prefrontal cortex and hippocampus. We then tested whether different doses (1 and 3 mg/kg) of a CB1R antagonist, Rimonabant, modify binge-like intake or the development of a conditioned place preference (CPP) to sucrose. CB1R blockade reduced binge-like intake of sucrose and blocked a sucrose CPP, but only in rats that had undergone 28 days of sucrose consumption. These findings indicate that sucrose bingeing alters the ECS in reward-related areas, modifications that exacerbate the effect of CB1R blockade on sucrose reward. Overall, our results broaden the understanding of neural alterations associated with bingeing eating and demonstrate an important role for CB1R mechanisms in reward processing. In addition, these findings have implications for understanding substance abuse, which is also characterized by excessive and maladaptive intake, pointing towards addictive-like properties of palatable food.

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.

Supplemental Digital Content is Available in the Text. Mefloquine/amitriptyline THN101 combination reduces cuff-induced neuropathic hypersensitivity by recruiting noradrenergic descending pathways and α2 adrenoceptors and by blocking connexin in mice., Tricyclic antidepressants that inhibit serotonin and noradrenaline reuptake, such as amitriptyline, are among the first-line treatments for neuropathic pain, which is caused by a lesion or disease affecting the somatosensory nervous system. These treatments are, however, partially efficient to alleviate neuropathic pain symptoms, and better treatments are still highly required. Interactions between neurons and glial cells participate in neuropathic pain processes, and importantly, connexins—transmembrane proteins involved in cell–cell communication—contribute to these interactions. In a neuropathic pain model in rats, mefloquine, a connexin inhibitor, has been shown to potentiate the antihyperalgesic effect of amitriptyline, a widely used antidepressant. In this study, we further investigated this improvement of amitriptyline action by mefloquine, using the cuff model of neuropathic pain in mice. We first observed that oral mefloquine co-treatment prolonged the effect of amitriptyline on mechanical hypersensitivity by 12 hours after administration. In addition, we showed that this potentiation was not due to pharmacokinetic interactions between the 2 drugs. Besides, lesional and pharmacological approaches showed that the prolonged effect was induced through noradrenergic descending pathways and the recruitment of α2 adrenoceptors. Another connexin blocker, carbenoxolone, also improved amitriptyline action. Additional in vitro studies suggested that mefloquine may also directly act on serotonin transporters and on adenosine A1 and A2A receptors, but drugs acting on these other targets failed to amplify amitriptyline action. Together, our data indicate that pharmacological blockade of connexins potentiates the therapeutic effect of amitriptyline in neuropathic pain.

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.

In rodents, morphine analgesia is influenced by sex. However, conflicting results exist regarding the interaction between sex and morphine analgesic tolerance. Morphine is metabolized in the liver and brain into morphine-3-glucuronide (M3G). Sex differences in morphine metabolism and differential metabolic adaptations during tolerance development might explain the behavioral discrepancies. The present article investigates the differences in peripheral and central morphine metabolism after acute and chronic morphine treatment in male and female mice.The first experiment aimed to determine whether morphine analgesia and tolerance differ between male and female mice using the tail-immersion test. The second experiment evaluated morphine and M3G metabolic kinetics in the blood using LC-MS/MS. Morphine and M3G were also quantified in several central nervous system (CNS) regions after acute and chronic morphine treatment. Finally, the blood-brain barrier permeability of M3G was assessed in male and female mice.This study demonstrated that female mice showed weaker morphine analgesia. In addition, tolerance appeared earlier in females but the sex discrepancies observed seemed to be due to the initial differences in morphine analgesia rather than to sex-specific mechanisms involving metabolism. Additionally, compared to male mice, female mice showed higher levels of M3G in the blood and in several CNS regions, whereas lower levels of morphine were observed in these brain regions. These differences are attributable mainly to morphine central metabolism, which differed between males and females in pain-related brain regions, consistent with the weaker analgesic effect in females. However, the role of morphine metabolism in analgesic tolerance seems rather limited.

International audience; Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans‐Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA‐binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD‐generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

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.

International audience; Seasonal affective disorder (SAD), beyond mood changes, is characterized by alterations in daily rhythms of behavior and physiology. The pathophysiological conditions of SAD involve changes in day length and its first-line treatment is bright light therapy. Animal models using nocturnal rodents have been studied to elucidate the neurobiological mechanisms of depression, but might be ill suited to study the therapeutic effects of light in SAD since they exhibit light-aversive responses. Here Arvicanthis ansorgei, a diurnal rodent, was used to determine behavioral, molecular and brain dopamine changes in response to exposure to a winter-like photoperiod consisting of a light-dark cycle with 8 h of light, under diminished light intensity, and 16 h of darkness. Furthermore, we evaluated whether timed-daily bright light exposure has an effect on behavior and brain physiology of winter-like exposed animals. Arvicanthis under a winter-like condition showed alterations in the synchronization of the locomotor activity rhythm to the light-dark cycle. Moreover, alterations in day-night activity of dopaminergic neurotransmission were revealed in the nucleus accumbens and the dorsal striatum, and in the day-night clock gene expression in the suprachiasmatic nucleus. Interestingly, whereas dopamine disturbances were reversed in animals exposed to daily light at early or late day, altered phase of the daily rhythm of locomotion was reverted only in animals exposed to light at the late day. Moreover, Per2 gene expression in the SCN was also affected by light exposure at late day in winter-like exposed animals. These findings suggest that light induces effects on behavior by mechanisms that rely on both circadian and rhythm-independent pathways influencing the dopaminergic circuitry. This last point might be crucial for understanding the mechanisms of non-pharmacological treatment in SAD.

Circadian rhythms in nocturnal and diurnal mammals are primarily synchronized to local time by the light/dark cycle. However, nonphotic factors, such as behavioral arousal and metabolic cues, can also phase shift the master clock in the suprachiasmatic nuclei (SCNs) and/or reduce the synchronizing effects of light in nocturnal rodents. In diurnal rodents, the role of arousal or insufficient sleep in these functions is still poorly understood. In the present study, diurnal Sudanian grass rats,Arvicanthis ansorgei, were aroused at night by sleep deprivation (gentle handling) or caffeine treatment that both prevented sleep. Phase shifts of locomotor activity were analyzed in grass rats transferred from a light/dark cycle to constant darkness and aroused in early night or late night. Early night, but not late night, sleep deprivation induced a significant phase shift. Caffeine on its own induced no phase shifts. Both sleep deprivation and caffeine treatment potentiated light-induced phase delays and phase advances in response to a 30 min light pulse, respectively. Sleep deprivation in early night, but not late night, potentiated light-induced c-Fos expression in the ventral SCN. Caffeine treatment in midnight triggered c-Fos expression in dorsal SCN. Both sleep deprivation and caffeine treatment potentiated light-induced c-Fos expression in calbindin-containing cells of the ventral SCN in early and late night. These findings indicate that, in contrast to nocturnal rodents, behavioral arousal induced either by sleep deprivation or caffeine during the sleeping period potentiates light resetting of the master circadian clock in diurnal rodents, and activation of calbindin-containing suprachiasmatic cells may be involved in this effect.SIGNIFICANCE STATEMENTArousing stimuli have the ability to regulate circadian rhythms in mammals. Behavioral arousal in the sleeping period phase shifts the master clock in the suprachiasmatic nuclei and/or slows down the photic entrainment in nocturnal animals. How these stimuli act in diurnal species remains to be established. Our study in a diurnal rodent, the Grass rat, indicates that sleep deprivation in the early rest period induces phase delays of circadian locomotor activity rhythm. Contrary to nocturnal rodents, both sleep deprivation and caffeine-induced arousal potentiate the photic entrainment in a diurnal rodent. Such enhanced light-induced circadian responses could be relevant for developing chronotherapeutic strategies.

In the published article, there was an error in affiliation "2." Instead of "Global Drug Discovery, Global Therapeutic Research Groups, Gynecological Therapies, Bayer Healthcare, Berlin, Germany", it should be "Laboratoire de Spectrometrie deMasse BioOrganique, IPHC-DSA, CNRS UMR7178 and Universite de Strasbourg, Strasbourg, France." Additionally, the current address of one author has been added as a footnote and is denoted using. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram. ispartof: NEURON vol:103 issue:1 pages:133-+ ispartof: location:United States status: published

International audience; Background and purpose: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. 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 question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels.Experimental approach: Male C57BL/6 mice were injected with morphine or saline solution for eight 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 2 H; 15%, w/w). Mice were then killed 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 forms.Key results: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study.Conclusions and implications: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.

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.

In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. Despite the widespread use of these drugs, the mechanism underlying their therapeutic action in this pain context remains partly elusive. The present study combined data collected in male and female mice from a model of neuropathic pain and data from the clinical setting to understand how antidepressant drugs act. We show two distinct mechanisms by which the selective inhibitor of serotonin and noradrenaline reuptake duloxetine and the tricyclic antidepressant amitriptyline relieve neuropathic allodynia. One of these mechanisms is acute, central, and requires descending noradrenergic inhibitory controls and α2Aadrenoceptors, as well as the mu and delta opioid receptors. The second mechanism is delayed, peripheral, and requires noradrenaline from peripheral sympathetic endings and β2adrenoceptors, as well as the delta opioid receptors. We then conducted a transcriptomic analysis in dorsal root ganglia, which suggested that the peripheral component of duloxetine action involves the inhibition of neuroimmune mechanisms accompanying nerve injury, including the downregulation of the TNF-α–NF-κB signaling pathway. Accordingly, immunotherapies against either TNF-α or Toll-like receptor 2 (TLR2) provided allodynia relief. We also compared duloxetine plasma levels in the animal model and in patients and we observed that patients' drug concentrations were compatible with those measured in animals under chronic treatment involving the peripheral mechanism. Our study highlights a peripheral neuroimmune component of antidepressant drugs that is relevant to their delayed therapeutic action against neuropathic pain.SIGNIFICANCE STATEMENTIn addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. However, the mechanism by which antidepressant drugs can relieve neuropathic pain remained in part elusive. Indeed, preclinical studies led to contradictions concerning the anatomical and molecular substrates of this action. In the present work, we overcame these apparent contradictions by highlighting the existence of two independent mechanisms. One is rapid and centrally mediated by descending controls from the brain to the spinal cord and the other is delayed, peripheral, and relies on the anti-neuroimmune action of chronic antidepressant treatment.

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.

Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

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.

International audience; Morphine derived from Papaver somniferum is commonly used as an analgesic compound for pain relief. It is now accepted that endogenous morphine, structurally identical to vegetal morphine-alkaloid, is synthesized by mammalian cells from dopamine. Morphine binds mu opioid receptor and induces antinociceptive effects. However, the exact role of these compounds is a matter of debate although different links with infection, sepsis, inflammation, as well as major neurological pathologies (Parkinson's disease, schizophrenia) have been proposed. The present review describes endogenous morphine and morphine derivative discovery, synthesis, localization and potential implications in physiological and pathological processes.

Interactions between the various cell types that constitute a solid tumour are essential to the biology of the tumour. We evaluated the effect of morphine on the proangiogenic interaction taking place between macrophages and breast cancer cells in vitro. The conditioned medium (CM) from breast cancer cells co-cultured with macrophages elicited endothelial cell proliferation and tube formation. This effect was inhibited if the co-culture occurred in the presence of morphine. The CM from breast cancer cells or macrophages grown individually, whether or not prepared in the presence of morphine, was ineffective in stimulating EC proliferation or tube formation. Using a mouse antibody array, we identified several angiogenesis-regulating factors differentially expressed in the CM of co-cultured cells prepared in the presence or absence of morphine, amongst which interleukin (IL)-6, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A. VEGF was induced in both cell types by the co-culture and this was prevented by morphine in a non-naloxone reversible fashion. The effect of CM from co-cultured cells on endothelial tube formation, but not proliferation, was prevented by anti-VEGF neutralizing antibody. Our results indicate that morphine prevents, in part via modulating VEGF-A expression, the pro-angiogenic interaction between macrophages and breast cancer cells.

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.

Depuis le debut des annees 1980, la morphine endogene dont la structure est identique a la morphine produite par le pavot a ete caracterisee dans de nombreuses cellules et tissus de mammiferes. Un nombre croissant de publications montre sa presence et suggere son implication dans differents processus, a la fois aux niveaux central et peripherique.

Interactions between cancer cells and stromal cells in the tumour microenvironment play a key role in the control of invasiveness, metastasis and angiogenesis. Macrophages display a range of activation states in specific pathological contexts and alternatively activated (M2) macrophages can promote tumour aggressiveness. Opioids are able to modulate tumour growth and metastasis. We tested whether morphine modulates the activation of macrophages induced by (i) interleukin-4 (IL-4), the prototypical M2 polarization-inducing cytokine, or (ii) coculture with breast cancer cells. We showed that IL-4 causes increased MMP-9 production and expression of the alternative activation markers arginase-1 and MRC-1. Morphine prevented IL-4-induced increase in MMP-9 in a naloxone- and methylnaltrexone-reversible fashion. Morphine also prevented IL-4-elicited alternative activation of RAW264.7 macrophages. Expression of MMP-9 and arginase-1 were increased when RAW264.7 were subjected to paracrine activation by 4T1 cells and this effect was prevented by morphine via an opioid receptor-mediated mechanism. Morphine further decreased 4T1 breast cancer cell invasion elicited by co-culture with RAW264.7. Reduction of MMP-9 expression and alternative activation of macrophages by morphine was confirmed using mouse bone marrow-derived macrophages. Taken together, our results indicate that morphine may modulate tumour aggressiveness by regulating macrophage protease production and M2 polarization within the tumour microenvironment.

Secretory granules of chromaffin cells from the adrenal medulla store catecholamines and a variety of peptides that are secreted in the extracellular medium during exocytosis. Among these fragments, several natural peptides displaying antimicrobial activities at the micromolar range have been isolated and characterized. We have shown that these peptides, derived from the natural processing of chromogranins (CGs), proenkephalin-A (PEA) and free ubiquitin (Ub), are released into the circulation and display antibacterial and antifungal activities. In this review we focus on three naturally secreted antimicrobial peptides corresponding to CGA1-76 (vasostatin-I), the bisphosphorylated form of PEA209-237 (enkelytin) and Ub. In addition, the antimicrobial properties of the synthetic active domains of vasostatin-I (CGA47-66 or chromofungin) and Ub (Ub65-76 or ubifungin) are reported.

The secretory granules from adrenal medullary chromaffin cells contain a complex mixture of low-molecular mass constituents such as catecholamines, ascorbate, nucleotides, calcium, peptides, and several high-molecular mass water-soluble proteins including chromogranins and proenkephalin-A. These proteins are sequestered into secretory granules in which processing yields a large variety of peptides. These fragments are released into the extracellular space upon cell stimulation and are recovered in blood, lymph, cerebrospinal fluid, and synovial fluid. Some of them have biological activity on cells in an autocrine, paracrine, or endocrine fashion. In addition, we have shown that peptides with antimicrobial activity are present with the secretory chromaffin granules and demonstrated that they are released from stimulated chromaffin cells. We have shown that posttranslational modifications modulate the antimicrobial activities. For some peptides, using confocal laser microscopy, we have examined the interaction of the rhodaminated peptides with biological membranes. In addition, we have shown that chromofungin, the antifungal peptide corresponding to chromogranin A(47-66), can bind calmodulin in the presence of calcium and induce inhibition of calcineurin, a calmodulin-dependent enzyme. Because these antibacterial peptides are colocalized with catecholamines, they may be activated during stress, playing a role as a first protective barrier against bacterial infection, and thus act as factors of the innate immunity shortly after infection and before the induction and mobilization of an adaptative immune system.

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.

The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders. Electronic supplementary material The online version of this article (doi:10.1007/s00429-014-0745-5) contains supplementary material, which is available to authorized users.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Mytilus edulis pedal ganglia at a level of 2.67+/-0.44 and 0.98+/-0.14 ng/ganglia, respectively by high performance liquid chromatography coupled to electrochemical detection. These opiate alkaloids were further identified by both gas-chromatography mass spectrometry and nanoflow electrospray ionization double quadrupole orthogonal acceleration Time of Flight mass spectrometry. In animals that were starved, the morphine level rose to 6.38+/-0.88 ng/ganglion and the morphine 6-glucoronide rose to a level of 23.0+/-3.2 ng/ganglion after 30 days. These studies demonstrate that opiate alkaloids are present as naturally occurring signal molecules whose levels respond to stress, i.e., starvation. Opiate alkaloids were not found in the animal's incubation media or food, demonstrating their synthesis occurred in the respective tissue. These new method of opiate alkaloid detection, conclusively proves that morphine and morphine-6-glucuronide are present in animal tissues.

Using nano-electrospray ionization double quadrupole orthogonal-acceleration time-of-flight mass spectrometry, we were able to detect sub-nanogram levels of endogenous morphine and its metabolite morphine-6-glucuronide in the rat pheochromocytoma cell line. Furthermore, fragmentation of endogenous morphine-6-glucuronide (462.14 Da) by collision-induced dissociation yields morphine (286.14 Da) obtained from the cell samples and with the authentic material, further demonstrating the presence of endogenous morphine. The incubation medium was devoid of this material. This methodology represents a sensitive and accurate method to detect trace amounts of endogenous opiates at subnanogram levels in biological samples.

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63–70 and PEA224–237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209–237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.

Background: Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response. Endogenous morphine production has previously been demonstrated in humans after cardiac surgery with CPB. It has been hypothesized that morphine plays a role as an anti-inflammatory mediator in the systemic inflammatory response. The aim of this study was to investigate if the CPB procedure in itself elicits an endogenous morphine production in neonatal pigs. Methods: Endogenous morphine production was measured in arterial blood in piglets exposed to sternotomy alone (sham group, n=10) or sternotomy and CPB (n=10). Blood samples were obtained immediately after the induction of anaesthesia, at the end of CPB and 4 h later. Morphine in arterial blood was detected by radioimmunoassay and confirmed by gas chromatography mass spectrometry. Results: Animals undergoing CPB showed detectable endogenous morphine concentrations immediately after CPB, with increased concentrations postoperatively. There was no measurable morphine production in the sham operated pigs. Conclusion: The CPB procedures elicits an endogenous morphine production in neonatal pigs. This morphine response is analogous to the previously demonstrated response in patients subjected to cardiac surgery and CPB.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.