Altered reward processing following sucrose bingeing in male and female mice.

Objectives: Binge eating disorder (BED) is the most prevalent eating disorder associated with multiple adverse health effects, especially mental health issues, including substance use disorders and mood and anxiety disorders. Given these high comorbidities, the objective of our study was to examine whether bingeing behavior would lead to altered perception of reinforcing properties of EtOH and changes in well-being. Methods: We used a sucrose bingeing model based on an intermittent access paradigm with a two-bottle choice, without fasting, in male and female mice. We examined the effect of 2-week sucrose paradigm on ethanol-reinforcing properties using a conditioned place preference test (CPP). Well-being, anxiety- and depressive-like behavioral tests were performed to assess emotional state following 2 and 8-week sucrose bingeing paradigm. Results: Mice with intermittent access to sucrose developed a binge-like behavior assessed by higher sucrose intake and escalation rate during the 1st hour of access, in comparison with mice with a continuous sucrose access. We show for the first time that sucrose bingeing in mice modifies positive reinforcing effect of EtOH in a CPP paradigm without marked alteration of emotional state. Interestingly, prolonging sucrose access for 8 weeks revealed an exacerbated bingeing behavior in female mice, and some signs of emotional state alterations in female with continuous access. Discussion: In sum, our findings broaden the understanding of behavioral alterations associated with bingeing, highlighting the need to investigate addictive-like properties of palatable food both in male and female mice.

The Role of the Endocannabinoid System in Binge Eating Disorder.

Eating disorders are multifactorial disorders that involve maladaptive feeding behaviors. Binge eating disorder (BED), the most prevalent of these in both men and women, is characterized by recurrent episodes of eating large amounts of food in a short period of time, with a subjective loss of control over eating behavior. BED modulates the brain reward circuit in humans and animal models, which involves the dynamic regulation of the dopamine circuitry. The endocannabinoid system plays a major role in the regulation of food intake, both centrally and in the periphery. Pharmacological approaches together with research using genetically modified animals have strongly highlighted a predominant role of the endocannabinoid system in feeding behaviors, with the specific modulation of addictive-like eating behaviors. The purpose of the present review is to summarize our current knowledge on the neurobiology of BED in humans and animal models and to highlight the specific role of the endocannabinoid system in the development and maintenance of BED. A proposed model for a better understanding of the underlying mechanisms involving the endocannabinoid system is discussed. Future research will be necessary to develop more specific treatment strategies to reduce BED symptoms.

Hippocampal mu opioid receptors are modulated following cocaine self-administration in rat.

Cocaine addiction is a complex pathology induced by long-term brain changes. Understanding the neurochemical changes underlying the reinforcing effects of this drug of abuse is critical for reducing the societal burden of drug addiction. The mu opioid receptor plays a major role in drug reward. This receptor is modulated by chronic cocaine treatment in specific brain structures, but few studies investigated neurochemical adaptations induced by voluntary cocaine intake. In this study, we investigated whether intravenous cocaine-self administration (0.33 mg/kg/injection, fixed-ratio 1 [FR1], 10 days) in rats induces transcriptional and functional changes of the mu opioid receptor in reward-related brain regions. Epigenetic processes with histone modifications were examined for two activating marks, H3K4Me3, and H3K27Ac. We found an increase of mu opioid receptor gene expression along with a potentiation of its functionality in hippocampus of cocaine self-administering animals compared to saline controls. Chromatin immunoprecipitation followed by qPCR revealed no modifications of the histone mark H3K4Me3 and H3K27Ac levels at mu opioid receptor promoter. Our study highlights the hippocampus as an important target to further investigate neuroadaptive processes leading to cocaine addiction.

The endocannabinoid system is modulated in reward and homeostatic brain regions following diet-induced obesity in rats: a cluster analysis approach.

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.

Binge sucrose-induced neuroadaptations: A focus on the endocannabinoid system.

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.

Deletion of mu opioid receptors reduces palatable solution intake in a mouse model of binge eating.

Binge eating in humans is driven by hedonic properties of food, suggesting that brain reward systems may contribute to this behaviour. We examined the role of mu opioid receptors (MOP) in binge eating by examining sweet solution intake in mice with genetic deletion of the MOP. Wildtype and MOP knockout mice had 4 hours access to food in the home cage combined with limited (4 hours) access to sucrose (17.1% w/v) or saccharin (0.09% w/v), or continuous (24 hours) access to sucrose. Only limited access groups exhibited binge intake, measured as increased solution consumption during the first hour. Knockout mice consumed less solution and food during the first hour as well as less food each day compared with wildtype mice. Limited access groups consumed more food and gained more weight than continuous access groups, and the effect was magnified in saccharin-consuming mice. Indeed, the increased food consumption in animals given limited access to saccharin was so excessive that caloric intake of this group was significantly higher than either of the sucrose groups (limited or continuous access). Within this group, females consumed more food per bodyweight than males, highlighting important sex differences in feeding behaviours under restricted access schedules.

The rostromedial tegmental nucleus gates fat overconsumption through ventral tegmental area output in male rats.

Excessive consumption of palatable foods that are rich in fats and sugars has contributed to the increasing prevalence of obesity worldwide. Similar to addictive drugs, such foods activate the brain's reward circuit, involving mesolimbic dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and the prefrontal cortex. Neuroadaptations occurring in this circuit are hypothesized to contribute to uncontrolled consumption of such foods, a common feature of most of eating disorders and obesity. The rostromedial tegmental nucleus (RMTg), also named tail of the VTA (tVTA), is an inhibitory structure projecting to the VTA and the lateral hypothalamus (LH), two key brain regions in food intake regulation. Prior research has demonstrated that the RMTg responds to addictive drugs and influences their impact on mesolimbic activity and reward-related behaviors. However, the role of the RMTg in food intake regulation remains largely unexplored. The present study aimed to investigate the role of the RMTg and its projections to the VTA and the LH in regulating food intake in rats. To do so, we examined eating patterns of rats with either bilateral excitotoxic lesions of the RMTg or specific lesions of RMTg-VTA and RMTg-LH pathways. Rats were exposed to a 6-week 'free choice high-fat and high-sugar' diet, followed by a 4-week palatable food forced abstinence and a 24 h re-access period. Our results indicate that an RMTg-VTA pathway lesion increases fat consumption following 6 weeks of diet and at time of re-access. The RMTg-LH pathway lesion produces a milder effect with a decrease in global calorie intake. These findings suggest that the RMTg influences palatable food consumption and relapse through its projections to the VTA.

Protracted abstinence from distinct drugs of abuse shows regulation of a common gene network.

Addiction is a chronic brain disorder. Prolonged abstinence from drugs of abuse involves dysphoria, high stress responsiveness and craving. The neurobiology of drug abstinence, however, is poorly understood. We previously identified a unique set of hundred mu-opioid receptor-dependent genes in the extended amygdala, a key site for hedonic and stress processing in the brain. Here we examined these candidate genes either immediately after chronic morphine, nicotine, Δ9-tetrahydrocannabinol or alcohol, or following 4 weeks of abstinence. Regulation patterns strongly differed among chronic groups. In contrast, gene regulations strikingly converged in the abstinent groups and revealed unforeseen common adaptations within a novel huntingtin-centered molecular network previously unreported in addiction research. This study demonstrates that, regardless the drug, a specific set of transcriptional regulations develops in the abstinent brain, which possibly contributes to the negative affect characterizing protracted abstinence. This transcriptional signature may represent a hallmark of drug abstinence and a unitary adaptive molecular mechanism in substance abuse disorders.

RSK2 signaling in brain habenula contributes to place aversion learning.

RSK2 is a Ser/Thr kinase acting in the Ras/MAPK pathway. Rsk2 gene deficiency leads to the Coffin-Lowry Syndrome, notably characterized by cognitive deficits. We found that mrsk2 knockout mice are unable to associate an aversive stimulus with context in a lithium-induced conditioned place aversion task requiring both high-order cognition and emotional processing. Virally mediated shRNA-RSK2 knockdown in the habenula, whose involvement in cognition is receiving increasing attention, also ablated contextual conditioning. RSK2 signaling in the habenula, therefore, is essential for this task. Our study reveals a novel role for RSK2 in cognitive processes and uncovers the critical implication of an intriguing brain structure in place aversion learning.

Hippocampal Cannabinoid 1 Receptors Are Modulated Following Cocaine Self-administration in Male Rats.

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.

LSP2-9166, an orthosteric mGlu4 and mGlu7 receptor agonist, reduces cocaine self-administration under a progressive ratio schedule in rats.

Cocaine addiction is a serious health issue in Western countries. Despite the regular increase in cocaine consumption across the population, there is no specific treatment for cocaine addiction. Critical roles for glutamate neurotransmission in the rewarding effects of psychostimulants as well as relapse have been suggested and accumulating evidence indicates that targeting mGlu group III receptors could represent a promising strategy to develop therapeutic compounds to treat addiction. In this context, the aim of our study was to examine the effect of LSP2-9166, a mGlu4/mGlu7 receptor orthosteric agonist, on the motivation for cocaine intake. We used an intravenous self-administration paradigm in male Wistar rats as a reliable model of voluntary drug intake. We first evaluated the direct impact of cocaine on Grm4 and Grm7 gene expression. Voluntary cocaine intake under a fixed ratio schedule of injections induced an increase of both mGlu4 and mGlu7 receptor transcripts in nucleus accumbens and hippocampus. We then evaluated the ability of LSP2-9166 to affect cocaine self-administration under a progressive ratio schedule of reinforcement. We found that this compound inhibits the motivation to obtain the drug, although it induced a hypolocomotor effect which could biais motivation index. Our findings demonstrate that mGlu group III receptors represent new targets for decreasing motivation to self-administer cocaine.

Can cocaine-induced neuroinflammation explain maladaptive cocaine-associated memories?

Persistent and intrusive memories define a number of psychiatric disorders, including posttraumatic stress disorder and substance use disorder. In the latter, memory for drug-paired cues plays a critical role in sustaining compulsive drug use as these are potent triggers of relapse. As with many drugs, cocaine-cue associated memory is strengthened across presentations as cues become reliable predictors of drug availability. Recently, the targeting of cocaine-associated memory through disruption of the reconsolidation process has emerged as a potential therapeutic strategy; reconsolidation reflects the active process by which memory is re-stabilized after retrieval. In addition, a separate line of work reveals that neuroinflammatory markers, regulated by cocaine intake, play a role in memory processes. Our review brings these two literatures together by summarizing recent findings on cocaine-associated reconsolidation and cocaine-induced neuroinflammation. We discuss the interactions between reconsolidation processes and neuroinflammation following cocaine use, concluding with a new perspective on treatment to decrease risk of relapse to cocaine use.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Neuroepigenetics and addictive behaviors: Where do we stand?

Substance use disorders involve long-term changes in the brain that lead to compulsive drug seeking, craving, and a high probability of relapse. Recent findings have highlighted the role of epigenetic regulations in controlling chromatin access and regulation of gene expression following exposure to drugs of abuse. In the present review, we focus on data investigating genome-wide epigenetic modifications in the brain of addicted patients or in rodent models exposed to drugs of abuse, with a particular focus on DNA methylation and histone modifications associated with transcriptional studies. We highlight critical factors for epigenomic studies in addiction. We discuss new findings related to psychostimulants, alcohol, opiate, nicotine and cannabinoids. We examine the possible transmission of these changes across generations. We highlight developing tools, specifically those that allow investigation of structural reorganization of the chromatin. These have the potential to increase our understanding of alteration of chromatin architecture at gene regulatory regions. Neuroepigenetic mechanisms involved in addictive behaviors could explain persistent phenotypic effects of drugs and, in particular, vulnerability to relapse.

CB1 Agonism Alters Addiction-Related Behaviors in Mice Lacking Mu or Delta Opioid Receptors.

Opioids are powerful analgesics but the clinical utility of these compounds is reduced by aversive outcomes, including the development of affective and substance use disorders. Opioid systems do not function in isolation so understanding how these interact with other neuropharmacological systems could lead to novel therapeutics that minimize withdrawal, tolerance, and emotional dysregulation. The cannabinoid system is an obvious candidate as anatomical, pharmacological, and behavioral studies point to opioid-cannabinoid interactions in the mediation of these processes. The aim of our study is to uncover the role of specific cannabinoid and opioid receptors in addiction-related behaviors, specifically nociception, withdrawal, anxiety, and depression. To do so, we tested the effects of a selective CB1 agonist, arachidonyl-2-chloroethylamide (ACEA), on mouse behavior in tail immersion, naloxone-precipitated withdrawal, light-dark, and splash tests. We examined cannabinoid-opioid interactions in these tests by comparing responses of wildtype (WT) mice to mutant lines lacking either Mu or Delta opioid receptors. ACEA, both acute or repeated injections, had no effect on nociceptive thresholds in WT or Mu knockout (KO) mice suggesting that analgesic properties of CB1 agonists may be restricted to chronic pain conditions. The opioid antagonist, naloxone, induced similar levels of withdrawal in all three genotypes following ACEA treatment, confirming an opioidergic contribution to cannabinoid withdrawal. Anxiety-like responses in the light-dark test were similar across WT and KO lines; neither acute nor repeated ACEA injections modified this behavior. Similarly, administration of the Delta opioid receptor antagonist, naltrindole, alone or in combination with ACEA, did not alter responses of WT mice in the light-dark test. Thus, there may be a dissociation in the effect of pharmacological blockade vs. genetic deletion of Delta opioid receptors on anxiety-like behavior in mice. Finally, our study revealed a biphasic effect of ACEA on depressive-like behavior in the splash test, with a prodepressive state induced by acute exposure, followed by a shift to an anti-depressive state with repeated injections. The initial pro-depressive effect of ACEA was absent in Mu KO mice. In sum, our findings confirm interactions between opioid and cannabinoid systems in withdrawal and reveal reduced depressive-like symptoms with repeated CB1 receptor activation.

Mu Opioid Receptors in Gamma-Aminobutyric Acidergic Forebrain Neurons Moderate Motivation for Heroin and Palatable Food.

BACKGROUND: Mu opioid receptors (MORs) are central to pain control, drug reward, and addictive behaviors, but underlying circuit mechanisms have been poorly explored by genetic approaches. Here we investigate the contribution of MORs expressed in gamma-aminobutyric acidergic forebrain neurons to major biological effects of opiates, and also challenge the canonical disinhibition model of opiate reward. METHODS: We used Dlx5/6-mediated recombination to create conditional Oprm1 mice in gamma-aminobutyric acidergic forebrain neurons. We characterized the genetic deletion by histology, electrophysiology, and microdialysis; probed neuronal activation by c-Fos immunohistochemistry and resting-state functional magnetic resonance imaging; and investigated main behavioral responses to opiates, including motivation to obtain heroin and palatable food. RESULTS: Mutant mice showed MOR transcript deletion mainly in the striatum. In the ventral tegmental area, local MOR activity was intact, and reduced activity was only observed at the level of striatonigral afferents. Heroin-induced neuronal activation was modified at both sites, and whole-brain functional networks were altered in live animals. Morphine analgesia was not altered, and neither was physical dependence to chronic morphine. In contrast, locomotor effects of heroin were abolished, and heroin-induced catalepsy was increased. Place preference to heroin was not modified, but remarkably, motivation to obtain heroin and palatable food was enhanced in operant self-administration procedures. CONCLUSIONS: Our study reveals dissociable MOR functions across mesocorticolimbic networks. Thus, beyond a well-established role in reward processing, operating at the level of local ventral tegmental area neurons, MORs also moderate motivation for appetitive stimuli within forebrain circuits that drive motivated behaviors.

Mu opioid receptor: a gateway to drug addiction.

Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders.

ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity.

Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.

Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization.

Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons and its contribution to central sensitization have been investigated. In adult rat spinal slice preparations, activation of C-fiber primary afferents by a brief exposure of capsaicin produces an eightfold to 10-fold increase in ERK phosphorylation (pERK) in superficial dorsal horn neurons. The pERK induction is reduced by blockade of NMDA, AMPA/kainate, group I metabotropic glutamate receptor, neurokinin-1, and tyrosine receptor kinase receptors. The ERK activation produced by capsaicin is totally suppressed by inhibition of either protein kinase A (PKA) or PKC. PKA or PKC activators either alone or more effectively together induce pERK in superficial dorsal horn neurons. Inhibition of calcium calmodulin-dependent kinase (CaMK) has no effect, but pERK is reduced by inhibition of the tyrosine kinase Src. The induction of cAMP response element binding protein phosphorylation (pCREB) in spinal cord slices in response to C-fiber stimulation is suppressed by preventing ERK activation with the MAP kinase kinase inhibitor 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) and by PKA, PKC, and CaMK inhibitors. Similar signaling contributes to pERK induction after electrical stimulation of dorsal root C-fibers. Intraplantar injection of capsaicin in an intact animal increases expression of pCREB, c-Fos, and prodynorphin in the superficial dorsal horn, changes that are prevented by intrathecal injection of PD98059. Intrathecal PD98059 also attenuates capsaicin-induced secondary mechanical allodynia, a pain behavior reflecting hypersensitivity of dorsal horn neurons (central sensitization). We postulate that activation of ionotropic and metabotropic receptors by C-fiber nociceptor afferents activates ERK via both PKA and PKC, and that this contributes to central sensitization through post-translational and CREB-mediated transcriptional regulation in dorsal horn neurons.

Peripheral axonal injury results in reduced mu opioid receptor pre- and post-synaptic action in the spinal cord.

In both the spared nerve injury (SNI) and spinal nerve ligation (SNL) rat peripheral neuropathic pain models the presynaptic inhibitory effect of the mu opioid receptor (MOR) agonist (DAMGO) on primary afferent-evoked excitatory postsynaptic currents (EPSCs) and miniature EPSCs in superficial dorsal horn neurons is substantially reduced, but only in those spinal cord segments innervated by injured primary afferents. The two nerve injury models also reduce the postsynaptic potassium channel opening action of DAMGO on lamina II spinal cord neurons, but again only in segments receiving injured afferent input. The inhibitory action of DAMGO on ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons is also reduced in affected segments following nerve injury. MOR expression decreases substantially in injured dorsal root ganglion neurons (DRG), while intact neighboring DRGs are unaffected. Decreased activation of MOR on injured primary afferent central terminals and the second order neurons they innervate may minimize any reduction by opioids of the spontaneous pain mediated by ectopic input from axotomized small diameter afferents. Retention of MOR expression and activity in nearby non-injured afferents will enable, however, an opioid-mediated reduction of stimulus-evoked and spontaneous pain carried by intact nociceptor afferents and we find that intrathecal DAMGO (1000 ng) reduces mechanical hypersensitivity in rats with SNL. Axotomy-induced changes in MOR may contribute to opioid- insensitive components of neuropathic pain while the absence of these changes in intact afferents may contribute to the opioid sensitive components.