Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Formation of chronic morphine withdrawal memories requires C1QL3-mediated regulation of PSD95 in the mouse basolateral amygdala.

Chronic morphine withdrawal memory formation is a complex process influenced by various molecular mechanisms. In this study, we aimed to investigate the contributions of the basolateral amygdala (BLA) and complement component 1, q subcomponent-like 3 (C1QL3), a secreted and presynaptically targeted protein, to the formation of chronic morphine (repeat dosing of morphine) withdrawal memory using conditioned place aversion (CPA) and chemogenetic methods. We conducted experiments involving the inhibition of the BLA during naloxone-induced withdrawal to assess its impact on CPA scores, providing insights into the significance of the BLA in the chronic morphine memory formation process. We also examined changes in C1ql3/C1QL3 expression within the BLA following conditioning. Immunofluorescence analysis revealed the colocalization of C1QL3 and the G protein-coupled receptor, brain-specific angiogenesis inhibitor 3 (BAI3) in the BLA, supporting their involvement in synaptic development. Moreover, we downregulated C1QL3 expression in the BLA to investigate its role in chronic morphine withdrawal memory formation. Our findings revealed that BLA inhibition during naloxone-induced withdrawal led to a significant reduction in CPA scores, confirming the critical role of the BLA in this memory process. Additionally, the upregulation of C1ql3 expression within the BLA postconditioning suggested its participation in withdrawal memory formation. The colocalization of C1QL3 and BAI3 in the BLA further supported their involvement in synaptic development. Furthermore, downregulation of C1QL3 in the BLA effectively hindered chronic morphine withdrawal memory formation, emphasizing its pivotal role in this process. Notably, we identified postsynaptic density protein 95 (PSD95) as a potential downstream effector of C1QL3 during chronic morphine withdrawal memory formation. Blocking PSD95 led to a significant reduction in the CPA score, and it appeared that C1QL3 modulated the ubiquitination-mediated degradation of PSD95, resulting in decreased PSD95 protein levels. This study underscores the importance of the BLA, C1QL3 and PSD95 in chronic morphine withdrawal memory formation. It provides valuable insights into the underlying molecular mechanisms, emphasizing their significance in this intricate process.

YTHDF1 in periaqueductal gray inhibitory neurons contributes to morphine withdrawal responses in mice.

BACKGROUND: Physical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear. METHODS: A naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal. RESULTS: Our results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses. CONCLUSIONS: YTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

Ketamine metabolite alleviates morphine withdrawal-induced anxiety via modulating nucleus accumbens parvalbumin neurons in male mice.

Opioid withdrawal generates extremely unpleasant physical symptoms and negative affective states. A rapid relief of opioid withdrawal-induced anxiety has obvious clinical relevance but has been rarely reported. We have shown that injection of ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) leads to a rapid alleviation of anxiety-like behaviors in male mice undergoing chronic morphine withdrawal. Here we investigated the contribution of nucleus accumbens shell (sNAc) parvalbumin (PV)-neurons to this process. Chronic morphine withdrawal was associated with higher intrinsic excitability of sNAc PV-neurons via reduced voltage-dependent potassium currents. Chemogenetic inhibition of sNAc PV-neurons reversed the enhanced excitability of PV-neurons and anxiety-like behaviors in these morphine withdrawal male mice, while activation of sNAc PV-neurons induced anxiety-like behaviors in naive male mice. (2R,6R)-HNK reversed the altered potassium currents and intrinsic excitability of sNAc PV-neurons. Our findings demonstrate an important contribution of sNAc PV-neurons to modulating morphine withdrawal-induced anxiety-like behaviors and rapid relief of anxiety-like behaviors by (2R,6R)-HNK, this newly identified target may have therapeutic potentials in treating opioid addiction and anxiety disorders.

Upregulation of dynorphin/kappa opioid receptor system in the dorsal hippocampus contributes to morphine withdrawal-induced place aversion.

Aversive emotion of opioid withdrawal generates motivational state leading to compulsive drug seeking and taking. Kappa opioid receptor (KOR) and its endogenous ligand dynorphin have been shown to participate in the regulation of aversive emotion. In the present study, we investigated the role of dynorphin/KOR system in the aversive emotion following opioid withdrawal in acute morphine-dependent mice. We found that blockade of KORs before pairing by intracerebroventricular injection of KOR antagonist norBNI (20, 40 µg) attenuated the development of morphine withdrawal-induced conditioned place aversion (CPA) behavior. We further found that morphine withdrawal increased dynorphin A expression in the dorsal hippocampus, but not in the amygdala, prefrontal cortex, nucleus accumbens, and thalamus. Microinjection of norBNI (20 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-induced CPA behavior. We further found that p38 MAPK was significantly activated in the dorsal hippocampus after morphine withdrawal, and the activation of p38 MAPK was blocked by pretreatment with norBNI. Accordingly, microinjection of p38 MAPK inhibitor SB203580 (5 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-produced CPA behavior. This study demonstrates that upregulation of dynorphin/KOR system in the dorsal hippocampus plays a critical role in the formation of aversive emotion associated with morphine withdrawal, suggesting that KOR antagonists may have therapeutic value for the treatment of opioid withdrawal-induced mood-related disorders.

Deficient mitochondrial respiration in astrocytes impairs trace fear conditioning and increases naloxone-precipitated aversion in morphine-dependent mice.

Mitochondria are abundant in the fine processes of astrocytes, however, potential roles for astrocyte mitochondria remain poorly understood. In the present study, we performed a systematic examination of the effects of abnormal oxidative phosphorylation in astrocytes on several mouse behaviors. Impaired astrocyte oxidative phosphorylation was produced by astrocyte-specific deletion of the nuclear mitochondrial gene, Cox10, that encodes an accessory protein of complex IV, the protoheme:heme-O-farnesyl transferase. As expected, conditional deletion of the Cox10 gene in mice (cKO mice) significantly reduced expression of COX10 and Cytochrome c oxidase subunit I (MTCO1) of Complex IV, resulting in decreased oxidative phosphorylation without significantly affecting glycolysis. No effects of the deletion were observed on locomotor activity, anxiety-like behavior, nociception, or spontaneous alternation. Cox10 cKO female mice exhibited mildly impaired novel object recognition, while Cox10 cKO male mice were moderately deficient in trace fear conditioning. No group-related changes were observed in conditional place preference (CPP) that assessed effects of morphine on reward. In contrast to CPP, Cox10 cKO mice demonstrated significantly increased aversive behaviors produced by naloxone-precipitated withdrawal following chronic exposure to morphine, that is, jumping and avoidance behavior as assessed by conditional place aversion (CPA). Our study suggests that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or aversive and stressful conditions.

The CRF1 receptor mediates social behavior deficits induced by opiate withdrawal.

Poor sociability and aggressive behavior are key clinical features of opioid use disorders. The corticotropin-releasing factor (CRF) system may mediate behavioral effects of substances of abuse but its implication in substance-induced social behavior deficits and outward-directed hostility remains largely unknown. CRF signaling is mediated by two receptor types, termed CRF1 and CRF2 . The present study aimed at understanding the role for the CRF1 receptor in social and aggressive behavior induced by withdrawal from repeated opiate administration. Thus, wild-type (CRF1 +/+), CRF1 receptor heterozygous (CRF1 +/-), and null mutant (CRF1 -/-) female and male mice were treated with saline or escalating doses of morphine (20-100 mg/kg, i.p.) during six consecutive days and tested in the three-chamber task for sociability (i.e., preference for an unfamiliar same-sex conspecific vs. an object) 7 days after the last administration. Moreover, aggressive biting behavior toward the unfamiliar conspecific was assessed during the three-chamber test. Opiate withdrawal disrupted sociability in CRF1 +/+ and CRF1 +/-, but not in CRF1 -/-, female mice, without affecting aggressive biting behavior in any genotype. In contrast, opiate withdrawal did not affect sociability but increased aggressive biting behavior in male mice, independently of CRF1 receptor-deficiency. Nevertheless, in opiate-withdrawn CRF1 +/+, but not CRF1 +/- and CRF1 -/-, male mice, sociability directly correlated with aggressive biting behavior, suggesting a role for the CRF1 receptor in hostility-linked social approach. These findings demonstrate the implication of the CRF1 receptor in social behavior deficits associated with repeated opiate administration and withdrawal, revealing a new potential target for the treatment of opioid use disorders.

Basolateral Amygdala SIRT1/PGC-1α Mitochondrial Biogenesis Pathway Mediates Morphine Withdrawal-Associated Anxiety in Mice.

BACKGROUND: Anxiety is a negative emotion that contributes to craving and relapse during drug withdrawal. Sirtuins 1 (SIRT1) has been reported to be critical in both negative emotions and drug addiction. However, it remains incompletely elucidated whether SIRT1 is involved in morphine withdrawal-associated anxiety. METHODS: We established a mouse model of anxiety-like behaviors induced by morphine withdrawal and then detected neuronal activity with immunofluorescence and mitochondrial morphology with electron microscopy, mitochondrial DNA contents with quantitative real-time PCR, and mitochondrial function with the ATP content detection kit and the Mitochondrial Complex IV Activity Kit in the basolateral amygdala (BLA). The mitochondrial molecules were detected by western blot. Then we used virus-mediated downregulation and overexpression of SIRT1 in BLA to investigate the effect of SIRT1 on anxiety and mitochondrial function. Finally, we examined the effects of pharmacological inhibition of SIRT1 on anxiety and mitochondrial function. RESULTS: We found that BLA neuronal activity, mitochondrial function, and mtDNA content were significantly higher in morphine withdrawal mice. Furthermore, the expression levels of mitochondrial molecules increased in BLA cells. Virus-mediated downregulation of SIRT1 in BLA prevented anxiety-like behaviors in morphine withdrawal mice, whereas overexpression of SIRT1 in BLA facilitated anxiety-like behaviors in untreated mice through the SIRT1/ peroxisome proliferator activated receptor gamma coactivator 1-alpha pathway. Intra-BLA infusion of selective SIRT1 antagonist EX527 effectively ameliorated anxiety-like behaviors and mitochondrial dysfunction in mice with morphine withdrawal. CONCLUSION: Our results implicate a causal role for SIRT1 in the regulation of anxiety through actions on mitochondrial biogenesis. Inhibitors targeting SIRT1 may have therapeutic potential for the treatment of opioid withdrawal-associated anxiety.

Astroglial Knockout of Glucocorticoid Receptor Attenuates Morphine Withdrawal Symptoms, but Not Antinociception and Tolerance in Mice.

The development of tolerance and drug dependence limit the clinical application of opioids for the treatment of severe pain. Glucocorticoid receptors (GRs) are among molecular substrates involved in these processes. Most studies focus on the role of neuronal GR, while the involvement of GR on glial cells is not fully understood. To address this issue, we used a transgenic model of conditional GR knockout mice, targeted to connexin 30-expressing astrocytes, treated with repeated doses of morphine. We observed no difference between control mice and astrocytic GR knockouts in the development of antinociceptive tolerance. Nevertheless, when animals were subjected to precipitated withdrawal, knockouts presented some attenuated symptoms, including jumping. Taken together, our data suggest that hippocampal and spinal astrocytic GRs appear to be involved in opioid withdrawal, and drugs targeting the GR may relieve some symptoms of morphine withdrawal without influencing its antinociceptive properties.

Chronic morphine intoxication reduces binding of HuD to BDNF long 3'-UTR, while morphine withdrawal stimulates BDNF expression in the frontal cortex of male Wistar rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) mediates opiate dependence phenomenon. In the brain of morphine dependent animals BDNF level is controlled transcriptionally, however, post-transcriptional mechanisms of BDNF regulation in this context remain unknown. Regulation of mRNA by binding of specific proteins to the 3'-untranslated region (3'-UTR) is one of such mechanisms. Among RNA-binding proteins neuronal Hu antigen D (HuD) is the best characterized positive regulator of BDNF, however its involvement in opiate dependence remains obscure. We suggested that HuD binding to the BDNF 3'-UTR may be linked to changes in BDNF expression induced by morphine. The aim of this study was to investigate potential association of HuD with BDNF 3'-UTR in relation to BDNF expression (Exon- and 3'-UTR-specific mRNA variants and protein level) in the frontal cortex and midbrain of male Wistar rats after chronic morphine intoxication and spontaneous withdrawal in dependent animals. RESULTS: After chronic morphine intoxication but not during morphine withdrawal HuD binding to the long BDNF 3'-UTR in the frontal cortex decreased as compared with the corresponding control group, however after intoxication BDNF expression did not change. The level of BDNF Exon I as well as mature BDNF polypeptide increased in the frontal cortex upon morphine withdrawal, while no changes in HuD binding could be detected. CONCLUSION: Thus, contrary to the assumption, HuD-BDNF 3'-UTR interaction and BDNF expression in the frontal cortex differentially change in a manner dependent on the context of morphine action.

Linagliptin, a Selective Dipeptidyl Peptidase-4 Inhibitor, Reduces Physical and Behavioral Effects of Morphine Withdrawal.

(1) Background: Recent data indicate that receptors for GLP-1 peptide are involved in the activity of the mesolimbic system. Thus, the purpose of the present study was to examine the effect of the selective dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on morphine dependence in mice. (2) Methods: Morphine dependence in mice was obtained by administration of increasing doses of morphine for eight consecutive days, twice a day. On the 9th day of the experiment, the naloxone-induced (2 mg/kg, ip) morphine withdrawal signs (jumping) were assessed. Moreover, behavioral effects of short-term (60 h after morphine discontinuation) and long-term (14 days after morphine discontinuation) morphine withdrawal were observed. In terms of behavioral effects, the depressive effect in the forced swim test and anxiety in the elevated plus maze test were investigated. Locomotor activity of mice was also studied. (3) Results: The administration of linagliptin (10 and 20 mg/kg, ip) for 8 consecutive days before morphine injections significantly diminished the number of naloxone-induced morphine withdrawal signs (jumping) in mice. In addition, the cessation of morphine administration induced depressive behavior in mice which were observed during short- and long-term morphine withdrawal. Linagliptin administered during morphine withdrawal significantly reduced the depressive behavior in studied mice. Furthermore, the short-term morphine withdrawal evoked anxiety which also was reduced by linagliptin in mice. (4) Conclusions: The present study reveals that GLP-1 receptors are involved in morphine dependence. What is more, linagliptin might be a valuable drug in attenuating the physical symptoms of morphine dependence. It might be also a useful drug in reducing emotional disturbances which may develop during the morphine withdrawal period.

The duration of withdrawal affects the muscarinic signaling in the nucleus accumbens after chronic morphine exposure in neonatal rats.

The infants experience withdrawal from opiates, and time-dependent adaptations in neuronal activity of nucleus accumbens (NAc) may be crucial for this process. A key adaptation is an increased release of acetylcholine. The present study investigates muscarinic acetylcholine receptors (mAChRs) functions in the NAc at short-term (SWT) and long-term (LWT) withdrawal time following chronic morphine exposure in neonatal rats. The inhibitory role of presynaptic mAChRs activation in spontaneous excitatory postsynaptic currents (sEPSCs) in medium spiny neurons was decreased at LWT but not at SWT. Whereas, the excitatory role of post/extrasynaptic mAChRs activation in membrane currents was reduced at LWT but enhanced at SWT. Furthermore, the inhibitory effect of acute morphine on post/extrasynaptic mAChRs-mediated inward currents was enhanced at SWT but not at LWT. These results suggest that withdrawal from morphine leads to downregulation of presynaptic and post/extrasynaptic mAChRs functions in the NAc, which may coregulate the development of withdrawal in neonates.NEW & NOTEWORTHY We investigated for the first time how the duration of withdrawal affects mAChRs functions in the nucleus accumbens in neonatal rats. Compared with short-term withdrawal time, rats showed downregulation of presynaptic and post/extrasynaptic mAChRs functions during long-term withdrawal time. Our finding introduces a new possible correlation between the mAChRs dysfunction in the nucleus accumbens and the development of withdrawal in neonates.

Projection from the basolateral amygdala to the anterior cingulate cortex facilitates the consolidation of long-term withdrawal memory.

The process through which early memories are transferred to the cerebral cortex to form long-term memories is referred to as memory consolidation, and the basolateral amygdala (BLA) is an important brain region involved in this process. Although functional connections between the BLA and multiple brain regions are critical for the consolidation of withdrawal memory, whether the projection from the BLA to the anterior cingulate cortex (ACC) is involved in the formation or consolidation of withdrawal memory remains unclear. In this paper, we used a chemical genetic method to specifically label the BLA-ACC projection in a combined morphine withdrawal and conditioned place aversion (CPA) animal model. We found that (1) the inhibition of the BLA-ACC projection during conditioning had no effects on the formation of early withdrawal memory; (2) the inhibition of the BLA-ACC projection had no effects on the retrieval of either early or long-term withdrawal memory; and (3) the persistent inhibition of the BLA-ACC projection after early withdrawal memory formation could inhibit the formation of long-term withdrawal memory and decrease Arc protein expression in the ACC. These results suggested that the persistent activation of the BLA-ACC projection after the formation of early withdrawal memory facilitates the formation of long-term withdrawal memory by increasing the plasticity of ACC neurons.

Role of glucocorticoids on noradrenergic and dopaminergic neurotransmission within the basolateral amygdala and dentate gyrus during morphine withdrawal place aversion.

Aversive memories related to drug withdrawal can generate a motivational state leading to compulsive drug taking. However, the mechanisms underlying the generation of these withdrawal memories remain unclear. Limbic structures, such as the basolateral amygdala (BLA) and the dentate gyrus (DG) of the hippocampus, play a crucial role in the negative affective component of morphine withdrawal. Given the prominent role of glucocorticoids (GCs), noradrenaline (NA), and dopamine (DA) in memory-related processes, in the present study, we employed the conditioned place aversion (CPA) paradigm to uncover the role of GCs on NA and DA neurotransmission within the BLA and NA neurotransmission within the DG during opiate-withdrawal conditioning (memory formation consolidation), and after reexposure to the conditioned environment (memory retrieval). We observed that adrenalectomy impaired naloxone-induced CPA. Memory retrieval was associated with an increase in dihydroxyphenylacetic acid (DOPAC) levels in the BLA in morphine-addicted animals in a GC-independent manner. Importantly, NA turnover was related with the expression of withdrawal physical signs during the conditioning phase and with locomotor activity during the test phase. On the other hand, reduced DA concentration in the BLA was correlated with the CPA score. Our results indicate that while noradrenergic system is more associated with the somatic consequences of withdrawal, dopaminergic neurotransmission modulates the affective state. Nevertheless, it seems necessary that both systems work together with GCs to enable aversive-memory formation and recall.

Venlafaxine inhibits naloxone-precipitated morphine withdrawal symptoms: Role of inflammatory cytokines and nitric oxide.

Opioid-induced neuroinflammation plays a role in the development of opioid physical dependence. Moreover, nitric oxide (NO) has been implicated in several oxidative and inflammatory pathologies. Here, we sought to determine whether treatment with venlafaxine during the development of morphine dependence could inhibit naloxone-precipitated withdrawal symptoms. The involvement of neuro-inflammation related cytokines, oxidative stress, and L-arginine (L-arg)-NO pathway in these effects were also investigated. Mice received morphine (50 mg/kg/daily; s.c.), plus venlafaxine (5 and 40 mg/kg, i.p.) once a day for 3 consecutive days. In order to evaluate the possible role of L-arg-NO on the effects caused by venlafaxine, animals received L-arg, L-NAME or aminoguanidine with venlafaxine (40 mg/kg, i.p.) 30 min before each morphine injection for 3 consecutive days. On 4th day of experiment, behavioral signs of morphine-induced physical dependence were evaluated after i.p. naloxone injection. Then, brain levels of tissue necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), brain-derived neurotrophic factor (BDNF), NO and oxidative stress factors including; total thiol, malondialdehyde (MDA) contents and glutathione peroxidase (GPx) activity were determined. Co-administration of venlafaxine (40 mg/kg) with morphine not only inhibited the naloxone-precipitated withdrawal signs including jumping and weight loss, but also reduced the up-regulation of TNF-α, IL-1ß, IL-6, NO and MDA contents in mice brain tissue. However, repeated administration of venlafaxine inhibited the decrease in the brain levels of BDNF, total thiol and GPx. Pre-administration of L-NAME and aminoguanidine improved, while L-arg antagonized the venlafaxine-induced effects. These results provide evidences that venlafaxine could be used as a candidate drug to inhibit morphine withdrawal through the involvement of inflammatory cytokines and l-arginine-NO in mice.

Cardiac Protective Role of Heat Shock Protein 27 in the Stress Induced by Drugs of Abuse.

Heat shock proteins (HSP) are induced after different stress situations. Some of these proteins, particularly HSP-27, function as markers to indicate cellular stress or damage and protect the heart during addictive processes. Morphine withdrawal induces an enhancement of sympathetic activity in parallel with an increased HSP-27 expression and phosphorylation, indicating a severe situation of stress. HSP-27 can interact with different intracellular signaling pathways. Propranolol and SL-327 were able to antagonize the activation of hypothalamic-pituitary adrenal (HPA) axis and the phosphorylation of HSP-27 observed during morphine withdrawal. Therefore, ß-adrenergic receptors and the extracellular signal-regulated kinase (ERK) pathway would be involved in HPA axis activity, and consequently, in HSP-27 activation. Finally, selective blockade of corticotrophin releasing factor (CRF)-1 receptor and the genetic deletion of CRF1 receptors antagonize cardiac adaptive changes. These changes are increased noradrenaline (NA) turnover, HPA axis activation and decreased HSP-27 expression and phosphorylation. This suggests a link between the HPA axis and HSP-27. On the other hand, morphine withdrawal increases µ-calpain expression, which in turn degrades cardiac troponin T (cTnT). This fact, together with a co-localization between cTnT and HSP-27, suggests that this chaperone avoids the degradation of cTnT by µ-calpain, correcting the cardiac contractility abnormalities observed during addictive processes. The aim of our research is to review the possible role of HSP-27 in the cardiac changes observed during morphine withdrawal and to understand the mechanisms implicated in its cardiac protective functions.

PKC inhibitor reversed the suppressive effect of orexin-A on IPSCs of locus coeruleus neurons in naloxone-induced morphine withdrawal.

The locus coeruleus (LC) as a target of addictive drugs receives a dense projection of orexinergic fibres from the lateral hypothalamus (LH) and is accordingly a candidate site for the expression of the somatic aspects of morphine withdrawal. Recently it has been shown that the inhibitory synaptic currents of LC neurons decrease partly through orexin type 1 receptors in the context of naloxone-induced morphine withdrawal; however, its cellular mechanism remains unclear. In this study, whole-cell patch clamp recordings of LC neurons in brainstem slices were used to investigate the impact of protein kinase C (PKC) on GABAergic inhibitory post-synaptic currents (IPSCs) in the context of naloxone-induced morphine withdrawal. Male Wistar rats (P14-P21) received morphine (20 mg/kg, i.p.) daily for 7 consecutive days to induce morphine dependency. Our results showed that the application of PKC inhibitor (Go 6983; 1 µM) alone did not decrease the probability of GABA release in the LC neurons of the morphine-treated rats in the presence of naloxone. Although, Go 6983 reversed the reduction of the amplitude of evoked IPSCs (eIPSCs) and spontaneous IPSCs (sIPSCs) frequency induced by orexin-A but did not change the sIPSCs amplitude. These results indicate that the suppressive effect of orexin-A on IPSCs is probably reversed by PKC inhibitor in the LC neurons of morphine-treated rats in the context of naloxone withdrawal.

Glucocorticoid receptor but not mineralocorticoid receptor mediates the activation of ERK pathway and CREB during morphine withdrawal.

Recent research suggests that glucocorticoids are involved in the development of addiction to drugs of abuse. They share this role with dopamine (DA), and with different signalling pathways and/or transcription factors such as extracellular-signal regulated kinases (ERK) and cAMP response element binding protein (CREB). However, the relation between them is not completely elucidated. In this report, we further characterize the role of glucocorticoid and mineralocorticoid receptor (GR and MR) signalling in DA turnover at the Nacc, and in opiate withdrawal-induced tyrosine hydroxylase (TH) expression, ERK and CREB phosphorylation (activation) in the nucleus of tractus solitarius (NTS-A2 ). The role of GR and MR signalling was assessed with the selective GR antagonist, mifepristone or the MR antagonist, spironolactone (i.p.). Rats were implanted two morphine (or placebo) pellets. Six days later rats were pretreated with mifepristone, spironolactone or vehicle 30 min before naloxone, and DA turnover, TH expression, ERK and CREB phosphorylation, were measured using HPLC and immunoblotting. Glucocorticoid receptor blockade attenuated ERK and CREB phosphorylation and the TH expression induced by morphine withdrawal. In contrast, no changes were seen after MR blockade. Finally, GR and MR blockade did not alter the morphine withdrawal-induced increase seen both in DA turnover and DA metabolite production, in the NAcc. These results show that not only ERK and CREB phosphorylation but also TH expression in the NTS is modulated by GR signalling. The present results suggest that GR is a therapeutic target to improve aversive events associated with opiate withdrawal.

Parvalbumin Interneurons of Central Amygdala Regulate the Negative Affective States and the Expression of Corticotrophin-Releasing Hormone During Morphine Withdrawal.

BACKGROUND: The central nucleus of the amygdala (CeA) is a crucial component of the neuronal circuitry mediating aversive emotion. Its role in the negative affective states during drug withdrawal includes changes in opioidergic, GABAergic, and corticotropin-releasing factor neurotransmission. However, the modulation of the neurobiological interconnectivity in the CeA and its effects in the negative reinforcement of drug dependents are poorly understood. METHOD: We performed electrophysiological recordings to assess the membrane excitability of parvalbumin (PV)+ interneurons in the CeA during chronic morphine withdrawal. We tested the morphine withdrawal-induced negative affective states, such as the aversive (assessed by conditioned place aversion), anxiety (assessed by elevated plus maze), and anhedonic-like (assessed by saccharin preference test) behaviors, as well as the mRNA level of corticotropin-releasing hormone (CRH) via optogenetic inhibition or activation of PV+ interneurons in the CeA. RESULT: Chronic morphine withdrawal increased the firing rate of CeA PV+ interneurons. Optogenetic inhibition of the activity of CeA PV+ interneurons attenuated the morphine withdrawal-induced negative affective states, such as the aversive, anxiety, and anhedonic-like behaviors, while direct activation of CeA PV+ interneurons could trigger those negative affective-like behaviors. Optogenetic inhibition of the CeA PV+ interneurons during the morphine withdrawal significantly attenuated the elevated CRH mRNA level in the CeA. CONCLUSION: The activity of PV+ interneurons in the CeA was up-regulated during chronic morphine withdrawal. The activation of PV+ interneurons during morphine withdrawal was crucial for the induction of the negative emotion and the up-regulation of CRH mRNA levels in the CeA.

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.