Juan-Antonio García-Carmona

The oxytocin analogue carbetocin prevents priming-induced reinstatement of morphine-seeking: Involvement of dopaminergic, noradrenergic and MOPr systems.

Relapse to illicit drug-seeking following abstinence is a major challenge for the treatment of addiction as no effective pharmacotherapy is available. We have recently shown that activating the central oxytocinergic system prevents emotional impairment and stress-induced reinstatement associated with opioid withdrawal. Here, we investigated whether the oxytocin analogue carbetocin (CBT) is able to reverse morphine-primed reinstatement of conditioned-place preference (CPP) in mice. The mechanism underlining the behavioural effect of CBT was investigated by assessing the involvement of the striatal noradrenergic and dopaminergic systems in CBT reversal of priming- and stress-induced reinstatement of opioid CPP. In addition, given recent evidence suggesting the presence of oxytocin receptor (OTR)-μ-opioid receptor (MOPr) interactions in the brain, we further explored these interactions by carrying out OTR autoradiographic binding in brain of mice lacking MOPr. CBT administration prevented priming-induced reinstatement of morphine CPP. While an acute effect of CBT in enhancing dopamine turnover was observed following stress- and priming-induced reinstatement, CBT significantly decreased striatal noradrenaline turnover only following priming-induced reinstatement. Moreover, a significant brain region- specific increase in OTR binding was observed in MOPr knockout mice, indicating the presence of a possible OTR-MOPr interaction, which may be involved in the modulation of relapse. These results support the oxytocinergic system as a promising target for the prevention of relapse to opioid use and highlight the differential involvement of monoaminergic systems on the effects of OTR stimulation in preventing stress- and priming-induced reinstatement of opioid CPP behaviour.

Role of Corticotropin-Releasing Factor (CRF) Receptor-1 on the Catecholaminergic Response to Morphine Withdrawal in the Nucleus Accumbens (NAc)

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine (DA) levels in brain regions receiving dense VTA input. Since the role of stress in drug addiction is well established, the present study examined the possible involvement of CRF1 receptor in the interaction between morphine withdrawal and catecholaminergic pathways in the reward system. The effects of naloxone-precipitated morphine withdrawal on signs of withdrawal, hypothalamo-pituitary-adrenocortical (HPA) axis activity, dopamine (DA) and noradrenaline (NA) turnover in the nucleus accumbens (NAc) and activation of VTA dopaminergic neurons, were investigated in rats pretreated with vehicle or CP-154,526 (selective CRF1R antagonist). CP-154,526 attenuated the increases in body weight loss and suppressed some of withdrawal signs. Pretreatment with CRF1 receptor antagonist resulted in no significant modification of the increased NA turnover at NAc or plasma corticosterone levels that were seen during morphine withdrawal. However, blockade of CRF1 receptor significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin (ACTH) levels, DA turnover and TH phosphorylation at Ser40 in the NAc. In addition, CP-154,526 reduced the number of TH containing neurons expressing c-Fos in the VTA after naloxone-precipitated morphine withdrawal. Altogether, these results support the idea that VTA dopaminergic neurons are activated in response to naloxone-precipitated morphine withdrawal and suggest that CRF1 receptors are involved in the activation of dopaminergic pathways which project to NAc.

Corticotropin-releasing factor (CRF) receptor-1 is involved in cardiac noradrenergic activity observed during naloxone-precipitated morphine withdrawal

The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [noradrenergic activity, induction of the hypothalamic–pituitary–adrenocortical (HPA) axis and activation of heat shock proteins (Hsps)]. Corticotropin‐releasing factor (CRF) pathways are important mediators in the negative symptoms of opioid withdrawal. We performed a series of experiments to characterize the role of the CRF1 receptor in the response of stress systems to morphine withdrawal and its effect in the heart using genetically engineered mice lacking functional CRF1 receptors.

Brain stress system response after morphine-conditioned place preference.

This study examined the involvement of the brain stress system in the reinforcing effects of morphine. One group of mice was conditioned to morphine using the conditioned place preference (CPP) paradigm and the other group received morphine in a home-cage (non-conditioned). Adrenocorticotropic hormone and corticosterone levels were measured by radioimmunoassay; phospho (p) CREB expression and the number of corticotropin-releasing factor (CRF) neurons and fibres were measured by immunohistochemistry in different brain areas. We observed that the number of CRF neurons in the paraventricular nucleus (PVN) was increased after morphine-induced CPP, which was paralleled with enhanced CRF-immunoreactivity fibres in the nucleus tractus solitarius (NTS) and ventral tegmental area (VTA) vs. home-cage group injected with morphine. Morphine exposure induced an increase in CREB phosphorylated at Ser133 in the PVN and central amygdale (CeA), whereas mice exhibiting morphine CPP had higher levels of pCREB in the PVN, CeA and bed nucleus of the stria terminalis (BNST). We also found that most of the CRF-positive neurons in the PVN, CeA and BNST co-express pCREB after morphine CPP expression, suggesting that the drug-associated environmental contexts can elicit neuronal activity in the brain stress system. From the present results it is clear that exposure to a drug-associated context remains a potent activator of signalling pathways leading to CRF activation in the brain stress system.

Accumbal dopamine, noradrenaline and serotonin activity after naloxone-conditioned place aversion in morphine-dependent mice.

Dopamine (DA) neurons not only show a pattern signaling the magnitude, delay and probability of rewards but also code negative motivation and aversive events. Beside DA, other systems such as noradrenaline (NA) and serotonin (5-HT) may also be implicated in naloxone-induced conditioned place aversion (CPA; an index of the aversive consequences of withdrawal). The purpose of the present study was to evaluate: (i) the turnover of DA, NA and 5-HT in the nucleus accumbens (NAc), one of the most important substrates for aversive states, (ii) the changes in tyrosine hydroxylase (TH) gene expression in the ventral tegmental area, and (iii) total TH protein levels and TH phosphorylation in the NAc after naloxone-induced morphine withdrawal. DA, NA and 5-HT turnover was evaluated by high-performance liquid chromatography (HPLC). TH gene expression was determined by real time quantitative PCR (RT-PCR) and total TH and TH phosphorylated at Ser31 and Ser40 were analyzed by Western blot. Present results show that the aversion for environmental cues paired with opioid withdrawal was higher than that observed in the saline group treated with naloxone, which indicates that morphine pretreatment potentiated the ability of naloxone to produce place aversion. In addition, present data show that naloxone-induced CPA positively correlated with an increase of DA and NA turnover in the NAc, which paralleled an increase in TH gene expression in the VTA and TH phosphorylation and enhanced TH protein levels in the NAc. Thus, the present study indicates that naloxone-induced aversion in morphine-dependent mice enhances DA and NA activity in the NAc and suggests that transcriptional and post-transcriptional regulation of TH could be involved in the hyperactivity of mesolimbic dopaminergic system observed in morphine-withdrawn mice.

CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors.

Sex Differences between CRF1 Receptor Deficient Mice following Naloxone-Precipitated Morphine Withdrawal in a Conditioned Place Aversion Paradigm: Implication of HPA Axis

Background Extinction period of positive affective memory of drug taking and negative affective memory of drug withdrawal, as well as the different response of men and women might be important for the clinical treatment of drug addiction. We investigate the role of corticotropin releasing factor receptor type one (CRF1R) and the different response of male and female mice in the expression and extinction of the aversive memory. Methodology/Principal Finding We used genetically engineered male and female mice lacking functional CRF1R. The animals were rendered dependent on morphine by intraperitoneally injection of increasing doses of morphine (10–60 mg/kg). Negative state associated with naloxone (1 mg/kg s.c.)-precipitated morphine withdrawal was examined by using conditioned place aversion (CPA) paradigm. No sex differences for CPA expression were found in wild-type (n = 29) or CRF1R knockout (KO) mice (n = 29). However, CRF1R KO mice presented less aversion score than wild-type mice, suggesting that CRF1R KO mice were less responsive than wild-type to continuous associations between drug administration and environmental stimuli. In addition, CPA extinction was delayed in wild-type and CRF1R KO male mice compared with females of both genotypes. The genetic disruption of the CRF1R pathway decreased the period of extinction in males and females suggesting that CRF/CRF1R is implicated in the duration of aversive memory. Our results also showed that the increase in adrenocorticotropic hormone (ACTH) levels observed in wild-type (n = 11) mice after CPA expression, were attenuated in CRF1R KO mice (n = 10). In addition, ACTH returned to the baseline levels in males and females once CPA extinction was finished. Conclusion/Significance These results suggest that, at least, CPA expression is partially due to an increase in plasma ACTH levels, through activation of CRF1R, which can return when CPA extinction is finished.

Sympathetic activity induced by naloxone-precipitated morphine withdrawal is blocked in genetically engineered mice lacking functional CRF1 receptor

There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction.

Role of CRF1 receptor in post-incisional plasma extravasation and nociceptive responses in mice

ABSTRACT The corticotropin‐releasing factor (CRF) is involved in a number of physiological functions including pain perception. The purpose of this study was to evaluate the role of CRF1 receptor in the long‐lasting post‐surgical changes in somatic nociceptive thresholds and in local inflammatory responses, using genetically engineered mice lacking functional CRF1 receptor. Animals underwent a plantar incision under anaesthesia with remifentanil (80 &mgr;g/kg s.c.) and sevoflurane. Mechanical thresholds (von Frey) and plasma extravasation (Evans blue) were evaluated at different time points. On postoperative day 20, mechanical thresholds had returned to baseline in CD1 mice (3.07 ± 6.21%), while B6,129CRHtklee mice presented significant hyperalgesia, which was similar in wild‐type (WT) (− 29.81 ± 8.89%) and CRF1 receptor knockout (KO) (− 37.10 ± 10.75%) mice, showing strain differences. The administration of naloxone (1 mg/kg, s.c.) on postoperative day 21 produced hyperalgesia revealing surgery‐induced latent pain sensitization. The extent of hyperalgesia was greater in KO versus WT mice, suggesting a role of CRF1 receptors in the upward modulation of endogenous opioid release. Furthermore, two days after surgery, plasma extravasation returned to baseline in WT mice but remained elevated in KO mice. In non‐manipulated B6,129CRHtklee KO mice we observed an increase in the number of writhes (41.25 ± 11.36) versus WT (23.80 ± 4.71), while in the tail immersion test no differences could be detected. Our results show that CRF/CRF1 receptors seem to be a protective role in latent pain sensitization induced by surgery and in the local inflammatory response to injury. HighlightsWe have used genetically engineered mice lacking CRF1 receptor to evaluate the role of this receptor in the long‐lasting post‐surgical changes in nociceptive thresholds and in local inflammatory responses.Our results suggest a role of CRF/CRF1 receptor in latent pain sensitization induced by surgery and in the inflammatory processes.The results of the present study may contribute to further understanding the mechanisms implicated in the latent pain sensitization and in the local inflammatory response to tissue injury.

Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.