Adductor Canal Nerve Block versus Intra-articular Anesthetic in Knee Arthroscopy: A Single-Blinded Prospective Randomized Trial.

Effective perioperative pain control following knee arthroscopy allows patients to reduce narcotic intake, avoid side effects of these medications, and recover more quickly. Adductor canal nerve blockade (ACB) and intra-articular injection of local anesthetic have been described as adjuvant treatments for postoperative pain control following surgery of the knee. This study directly compares the effect of each of these treatment modalities. Patients undergoing knee arthroscopy were blinded and randomized to receive either an ACB (n = 60) or intra-articular injection of local anesthetic (IAB, n = 64). Outcome measures included patient reported visual analog scale (VAS) scores at 1, 2, 4, 8, 16, 24, 36, 48 hours and 1 week and total narcotic consumption at 12, 24, and 48 hours postoperatively. Student's t-tests were used to compare unadjusted VAS scores at each time point and use of postoperative pain medication between treatment groups. Adjusted VAS scores were estimated in a multivariable general linear model with interaction of time and treatment group and other relevant covariates. There were no statistically significant differences between the two groups in terms of gender, age, body mass index, and insurance type. ACB patients had significantly higher pain scores than IAB patients at hours 1 and 2 (hour 1: 4.02 [2.99] vs. 2.59 [3.00], p = 0.009; hour 2: 3.12 [2.44] vs. 2.17 [2.62], p = 0.040). ACB patients had higher pain scores than IAB patients up to hour 16, though hours 4 to 16 were not significantly different. Adjusted covariate analyses demonstrate an additional statistically significant reduction in pain score in the IAB group at hour 4. There were no differences in narcotic consumption. Intraoperative local anesthetic and regional ACB each provides adequate pain control following knee arthroscopy, and intraoperative local anesthetic may provide enhanced pain control for up to 4 hours postoperatively. LEVEL OF EVIDENCE: : Level 1 evidence, randomized control trial.

Sex-differences in anxiety, neuroinflammatory markers, and enhanced fear learning following chronic heroin withdrawal.

Post-traumatic stress disorder (PTSD) and opioid use disorder (OUD) are comorbid in clinical populations. However, both pre-clinical and clinical studies of these co-occurring disorders have disproportionately represented male subjects, limiting the applicability of these findings. Our previous work has identified chronic escalating heroin administration and withdrawal can produce enhanced fear learning. This behavior is associated with an increase in dorsal hippocampal (DH) interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and glial fibrillary acidic protein (GFAP) immunoreactivity. Further, we have shown that these increases in IL-1ß and TNF-α are mechanistically necessary for the development of enhanced fear learning. Although these are exciting findings, this paradigm has only been studied in males. The current studies aim to examine sex differences in the behavioral and neuroimmune effects of chronic heroin withdrawal and future enhanced fear learning. In turn, we determined that chronic escalating heroin administration can produce withdrawal in female rats comparable to male rats. Subsequently, we examined the consequence of heroin withdrawal on future enhanced fear learning and IL-1ß, TNF-α, and GFAP immunoreactivity. Strikingly, we identified sex differences in these neuroimmune measures, as chronic heroin administration and withdrawal does not produce enhanced fear learning or immunoreactivity changes in females. Moreover, we determined whether heroin withdrawal produces short-term and long-term anxiety behaviors in both female and males. Collectively, these novel experiments are the first to test whether heroin withdrawal can sensitize future fear learning, produce neurobiological changes, and cause short-term and long-term anxiety behaviors in female rats.

Systemic administration of N-acetylcysteine during the extinction period and on the reinstatement day decreased the maintenance of morphine rewarding properties in the rats.

Many animal studies and early clinical trials suggested that N-acetylcysteine (NAC) may benefit addiction treatment. The present study tried to evaluate whether chronic administration of systemic NAC during the extinction period and acute administration of systemic NAC on the reinstatement day could reduce the maintenance of the morphine rewarding properties in the conditioned place preference (CPP) paradigm in the rats. Ninety-six adult male Wistar rats (190-220 g) were examined with morphine (7 mg/kg; sc) and saline (1 mL/kg; sc) during the 3-day conditioning phase in the CPP paradigm. After the acquisition of morphine CPP, different doses of NAC were daily administered during the extinction period (5, 10, 25, and 50 mg/kg; ip), or 30 min before the CPP test on the reinstatement day (2, 5, 10, 25, and 50 mg/kg; ip). Conditioning score and locomotor activity were recorded by the video tracking system and Ethovision software after acquisition on the post-conditioning day, the extinction period, and reinstatement day. Daily NAC administration in high doses (25 and 50 mg/kg; ip) reduced extinction-responding compared with the vehicle-control group during the extinction period. Although a single injection of NAC in doses 10, 25, 50 mg/kg decreased the reinstatement of morphine-induced CPP, two lower doses (2 and 5 mg/kg) could not significantly reduce the CPP scores. These are the first data suggesting that NAC's application during the extinction period could attenuate the morphine reward-associated behaviors in the rats. Moreover, NAC could inhibit the reinstatement of morphine CPP, which adds to the growing appreciation that the NAC may have potential therapeutic use in combating morphine dependence. It can be consistent with the hypothesis of the involvement of the glutamatergic system in the pathophysiology of addiction.

Effect of histone acetylation on maintenance and reinstatement of morphine-induced conditioned place preference and ΔFosB expression in the nucleus accumbens and prefrontal cortex of male rats.

Recently, epigenetic mechanisms are considered as the new potential targets for addiction treatment. This research was designed to explore the effect of histone acetylation on ΔFosB gene expression in morphine-induced conditioned place preference (CPP) in male rats. CPP was induced via morphine injection (5 mg/kg) for three consecutive days. Animals received low-dose theophylline (LDT) or Suberoylanilide Hydroxamic acid (SAHA), as an histone deacetylase (HDAC) activator or inhibitor, respectively, and a combination of both in subsequent extinction days. Following extinction, a priming dose of morphine (1 mg/kg) was administered to induce reinstatement. H4 acetylation and ΔFosB expression in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were assessed on the last day of extinction and the following CPP reinstatement. Our results demonstrated that daily administration of SAHA (25 mg/kg; i.p.), facilitated morphine-extinction and decreased CPP score in reinstatement of place preference. Conversely, injections of LDT (20 mg/kg; i.p.) prolonged extinction in animals. Co-administration of LDT and SAHA on extinction days counterbalanced each other, such that maintenance and reinstatement were no different than the control group. The gene expression of ΔFosB was increased by SAHA in NAc and mPFC compared to the control group. Administration of SAHA during extinction days, also altered histone acetylation in the NAc and mPFC on the last day of extinction, but not on reinstatement day. Collectively, administration of SAHA facilitated extinction and reduced reinstatement of morphine-induced CPP in rats. This study confirms the essential role of epigenetic mechanisms, specifically histone acetylation, in regulating drug-induced plasticity and seeking behaviors.

The effects of morphine, methadone, and fentanyl on mitochondria: A live cell imaging study.

The important role of mitochondria in maintaining normal brain cell function has been demonstrated in several neurodegenerative diseases where mitochondrial dysfunction is a prominent feature. Accumulating evidence indicates that opioids may induce neuronal cell death and inhibit neurogenesis, two factors that are dependent on normal mitochondrial function. The aim of the present study was to examine the effects of morphine, methadone, and fentanyl on MitoTracker-stained mitochondria. Cells from the neuroblastoma/glioma hybrid cell-line NG108-15 were seeded on 96-well cell culture plates and treated with MitoTracker for 30 min prior to opioid treatment. Morphine, methadone, and fentanyl were added at various concentrations and images of mitochondria were acquired every 30 min for four hours using a high-content imaging device. The parameters total mitochondrial area, mitochondrial network, as well as the number and mean area of mitochondrial objects were analyzed using automated image analysis. Methadone and fentanyl, but not morphine, decreased the mitochondrial network, the number of mitochondrial objects, and increased the mean area of mitochondrial objects. Both methadone and fentanyl altered mitochondrial morphology with no effects seen from morphine treatment. These data suggest that methadone and fentanyl impact mitochondrial morphology negatively, which may be associated with neuronal cell death.

The effects of morphine withdrawal and conditioned withdrawal on memory consolidation and c-Fos expression in the central amygdala.

The current study tested the hypothesis that drug withdrawal contributes to the addiction cycle in part because of an action on memory consolidation. Hence, four experiments in male Sprague-Dawley rats compared the effects of precipitated morphine withdrawal and conditioned morphine withdrawal on the consolidation of object memory and on activation of c-Fos in the central nucleus of the amygdala (CeA). It was found that immediate, but not 6 h delayed, post sample administration of 3 mg/kg of naltrexone significantly enhanced object memory in rats maintained, or previously maintained, on 10 mg/kg/day of morphine via osmotic minipumps. To establish whether conditioned withdrawal could also alter object memory, a contextual conditioning procedure was employed whereby morphine-maintained (10 mg/kg/day) animals received naltrexone (3 mg/kg) in a distinctive context (CS+) and vehicle in a separate context (CS-) for 10 days. During conditioning in the CS+, naltrexone suppressed locomotor activity, caused a rapid body weight loss and increased frequency of wet dog shakes. Interestingly, confinement to this CS+ immediately, but not 6 h, after the sample phase, also enhanced object memory. Finally, posttraining naltrexone and exposure to the CS+ both induced significant expression of c-Fos in the CeA. Therefore, this study reports for the first time that both acute precipitated withdrawal and conditioned withdrawal can facilitate memory consolidation, possibly through a common neural pathway that involves the central amygdala.

Suppressing effect of the novel positive allosteric modulator of the GABAB receptor, COR659, on locomotor hyperactivity induced by different drugs of abuse.

COR659 is a recently synthesized positive allosteric modulator (PAM) of the GABAB receptor. Similarly to all GABAB PAMs tested to date, COR659 has been reported to suppress different alcohol-related behaviors in rodents. The present study was designed to assess whether the anti-addictive properties of COR659 extend to drugs of abuse other than alcohol. Specifically, it investigated the effect of COR659 on cocaine-, amphetamine-, nicotine-, and morphine-induced locomotor hyperactivity in mice. To this aim, independent groups of CD1 mice were acutely pretreated with COR659 (0, 10, and 20 mg/kg; i.p.), then acutely treated with cocaine (0 and 10 mg/kg, s.c.), amphetamine (0 and 5 mg/kg; s.c.), nicotine (0 and 0.05 mg/kg; s.c.), or morphine (0 and 20 mg/kg; s.c.), and finally exposed for 60 min to a photocell-equipped motility cage. When given alone, both doses of COR659 were ineffective on spontaneous locomotor activity. Pretreatment with COR659 reduced, or even suppressed, the increase in motility counts induced by cocaine, amphetamine, nicotine, and morphine. Since locomotor hyperactivity is an attribute common to drugs of abuse, the results of the present study constitute the first line of evidence on the extension of the preclinical, anti-addictive profile of COR659 to cocaine, amphetamine, nicotine, and morphine.

Assessing the Role of Corticothalamic and Thalamo-Accumbens Projections in the Augmentation of Heroin Seeking in Chronically Food-Restricted Rats.

Drug addiction is a chronic disorder characterized by compulsive drug seeking, and involves repetitive cycles of compulsive drug use, abstinence, and relapse. In both human and animal models of addiction, chronic food restriction increases rates of relapse. Our laboratory has reported a robust increase in drug seeking following a period of withdrawal in chronically food-restricted rats compared with sated controls. Recently, we reported that activation of the paraventricular nucleus of the thalamus (PVT) abolished heroin seeking in chronically food-restricted rats. However, the precise inputs and outputs of the PVT that mediate this effect remain elusive. The goal of the current study was to determine the role of corticothalamic and thalamo-accumbens projections in the augmentation of heroin seeking induced by chronic food restriction. Male Long-Evans rats were trained to self-administer heroin for 10 d. Next, rats were removed from the self-administration chambers and were subjected to a 14 d withdrawal period while sated (unlimited access to food) or mildly food-restricted (FDR). On day 14, rats were returned to the self-administration context for a 3 h heroin-seeking test under extinction conditions during which corticothalamic and thalamo-accumbens neural activity was altered using chemogenetics. Surprisingly, chemogenetic activation or inhibition of corticothalamic projections did not alter heroin-seeking behavior. Chemogenetic activation of thalamo-accumbens shell, but not core, projectors attenuated heroin seeking in FDR rats. The results indicate an important role for the PVT to nucleus accumbens shell projections in the augmentation of heroin seeking induced by chronic food restriction.SIGNIFICANCE STATEMENT Relapse to heroin use is one of the major obstacles in the treatment of opiate addiction. Triggers for relapse are modulated by environmental challenges such as caloric restriction. Elucidating the brain mechanisms that underlie relapse is critical for evidence-based treatment development. Here we demonstrate a critical role for the input from the paraventricular thalamus (PVT), a hub for cortical, sensory, and limbic information, to the nucleus accumbens shell (an area known to be important for reward and motivation) in the augmentation of heroin seeking in food-restricted rats. Our findings highlight a previously unknown role for the PVT in heroin seeking following a period of abstinence.

Female rats self-administer heroin by vapor inhalation.

Over the last two decades the United States has experienced a significant increase in the medical and non-medical use of opioid drugs, resulting in record numbers of opioid-related overdoses and deaths. There was an initial increase in non-medical use of prescription opioids around 2002, followed later by increased heroin use and then most recently fentanyl. Inhalation is a common route of administration for opioids, with a documented history spanning back to Mediterranean antiquity and up through modern use with e-cigarette devices. Unfortunately, preclinical studies using inhalation as the route of administration remain relatively few. This study was conducted to determine the efficacy of e-cigarette vapor inhalation of heroin in rats. Non-contingent exposure to heroin or methadone vapor produced anti-nociceptive efficacy in male and female rats. Female rats were trained to self-administer heroin vapor; the most-preferring half of the distribution obtained more vapor reinforcers when the concentration of heroin was reduced in the vapor vehicle and when pre-treated with the opioid receptor antagonist naloxone. The anti-nociceptive effect of heroin self-administered by vapor was identical in magnitude to that produced by intravenous self-administration. Finally, anxiety-like behavior increased 24-48 h after last heroin vapor access, consistent with withdrawal signs observed after intravenous self-administration. In sum, these studies show that rewarding and anti-nociceptive effects of heroin are produced in rats by vapor inhalation using e-cigarette technology. Importantly, self-administration models by this route can be deployed to determine health effects of inhaled heroin or other opioids.

Reduction of anxiety level is associated with an oxidative-stress imbalance in the hippocampus in morphine administration period in male rats.

Hippocampus is a region of the brain that is famous for its role in memory. However recently it has been given great importance for a region that regulates emotion and anxiety. Alternation of anxiety level has been observed in drug abusers as a comorbid disorder. The aim of this study is to show that morphine by altering oxidative stress in the hippocampus causes anxiety level alternation. In this study 32 male Sprague-Dawley rats divided into four groups: Control, N-acetyl-cysteine-treated, morphine-treated and N-acetyl-cysteine + morphine-treated. After 14 days of morphine administration (5 mg/kg/rat/i.p.) and N-acetyl-cysteine administration (100 mg/kg/rat/i.p.), Anxiety was assessed with Elevated Plus Maze (EPM) after 14 days in all groups. Then rats were euthanized and the hippocampus was removed for assessing oxidative stress for malondialdehyde (MDA), glutathione and nitrite/nitrate. Our data showed that oxidative stress was disturbed in the hippocampus in morphine-treated rats. Malondialdehyde (MDA) increased and glutathione and nitrite/nitrate were reduced in morphine-treated rats compared to control and N-acetyl-cysteine-treated rats. N-Acetylcysteine treatment improved oxidative stress (OS) markers and anxiety. The anxiety that was assessed with Elevated Plus Maze, morphine-treated rats showed less anxiety level compared to N-acetyl-cysteine -treated and control rats. Morphine reduced the anxiety level. The reduction of anxiety level was associated with oxidative stress imbalance in the hippocampus. Thus, hippocampus can alter anxiety level.

Basolateral amygdala is required for reconsolidation updating of heroin-associated memory after prolonged withdrawal.

Postretrieval extinction procedures are effective nonpharmacological interventions for disrupting drug-associated memories. Nonetheless, the conditioned stimulus (CS) memory retrieval-extinction procedure is ineffective in inhibiting drug craving and relapse after prolonged withdrawal, which significantly undermines its therapeutic potential. In the present study, we showed that, unlike the CS memory retrieval-extinction procedure, noncontingent heroin injections (unconditioned stimulus [UCS]) 1 hour before the extinction sessions decreased the heroin-priming-induced reinstatement, renewal, and spontaneous recovery of heroin seeking after 28 days of withdrawal (ie, remote heroin-associated memories) in rats. The UCS retrieval manipulation induced reactivation of the basolateral amygdala (BLA) after prolonged withdrawal, and this reactivation was absent with the CS retrieval manipulation. Chemogenetic inactivation of the BLA abolished the inhibitory effect of the UCS memory retrieval-extinction procedure on heroin-priming-induced reinstatement after prolonged withdrawal. Furthermore, the combination of chemogenetic reactivation of BLA and CS retrieval-extinction procedure resembled the inhibitory effect of UCS retrieval-extinction procedure on heroin seeking after prolonged withdrawal. We also observed that the inhibitory effect of the UCS retrieval-extinction procedure is mediated by regulation of AMPA receptor endocytosis in the BLA. Our results demonstrate critical engagement of the BLA in reconsolidation updating of heroin-associated memory after prolonged withdrawal, extending our knowledge of the boundary conditions of the reconsolidation of drug-associated memories.

Low concentration of morphine protects against cell death, oxidative stress and calcium accumulation by nicotine in PC12 cells.

OBJECTIVE: Nicotine causes cell death in many cell lines. Morphine at low concentrations has protective effects against cell death. We investigated the effects of low concentration of morphine on nicotine-induced cell death in PC12 cells. MATERIALS AND METHODS: PC12 are cells that grow in DMEM culture medium. Cell viability was detected by MTT test and cells cytotoxicity was measured by LDH test. The activity of caspase-3 was diagnosed by the caspase activity colorimetric assay kit, and detection of mitochondrial membrane potential was confirmed by rhodamine 123 and TUNEL test was performed for DNA fragmentation detection. The fura-2 AM and also rhod 2-AM was used for measurement of intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+) m and finally, measurement of antioxidant enzyme activities was assessed. RESULTS: The low concentration of morphine increased cell viability and suppressed cell cytotoxicity, cell death and the formation of mitochondrial membrane potential compared to nicotine treated cells.  It also reduced the intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+)m concentration, respectively. CONCLUSION: Morphine as a pain reducer drug, in low concentrations, can protect PC12 cells from nicotine-induced cell death (Fig. 7, Ref. 59).

Transgenic increase in the ß-endorphin concentration in cerebrospinal fluid alleviates morphine-primed relapse behavior through the µ opioid receptor in rats.

BACKGROUND: Opioid-primed relapse is a global burden. Although current strategies have improved, optimal therapy is urgently needed. METHODS: A recombinant adenovirus (Ad-NEP) expressing ß-endorphin (ß-EP) was designed and injected intracerebroventricularly (icv) into the right lateral ventricle in rats. Spatial and temporal ß-EP expression in the lateral ventricle wall, subventricular zone and adjacent choroid plexus and the ß-EP concentration in the cerebrospinal fluid (CSF) were observed during a 21-day period. A morphine priming-induced conditioned place preference (CPP) rat model was established. The ß-EP-ir neuron counts, CSF ß-EP concentration, and CPP score, which were used to evaluate morphine-primed reinstatement following extinction, were recorded 7 days after the icv injection. Additionally, the rats were pretreated with the irreversible µ opioid receptor antagonist ß-funaltrexamine (ß-FNA) and the selective κ opioid receptor antagonist nor-binaltorphimine (nor-BNI) to identify the receptor-dependent mechanism. RESULTS: Both peak ß-EP expression in target neurons and the peak CSF ß-EP concentration occurred 7 to 8 days after Ad-NEP icv injection. The sustainable increase in the CSF ß-EP concentration was correlated with a decrease in the CPP score 7 days after the Ad-NEP icv injection. Furthermore, reinstatement was almost reversed by ß-FNA pretreatment 24 hours before the behavioral test, but nor-BNI had little effect. CONCLUSION: The increasing cerebrospinal fluid ß-endorphin concentrations showed that the therapeutic effect on opioid relapse occurred predominantly through a µ opioid receptor-dependent mechanism. The Ad-NEP adenovirus can be considered an alternative therapy for opioid relapse.

Enhanced Recovery Protocol for Laparoscopic Sleeve Gastrectomy: Are Narcotics Necessary?

BACKGROUND: Enhanced recovery after surgery (ERAS) protocols have improved patient experience and outcomes in a variety of fields, including bariatric surgery. Given the increasing opioid epidemic in the USA, we sought to determine the impact of our own ERAS protocol on narcotic usage following laparoscopic sleeve gastrectomy. METHODS: Retrospective chart review was performed on patients undergoing primary laparoscopic sleeve gastrectomy for 6 months before and after implementation of an ERAS protocol. Our protocol strongly discouraged the use of narcotics in the postoperative period. Specific outcomes of interest were postoperative narcotic usage, length of stay, complications, and readmissions. RESULTS: Patient characteristics were similar in the two groups. ERAS implementation did not correlate with changes in length of stay, complications, or readmissions. However, ERAS implementation was associated with dramatic reductions in the use of intravenous narcotics (100% vs 47%, p < 0.01) and oral schedule 2 narcotics (56% vs 6%, p < 0.01), with an increase in the usage of tramadol (0% vs 36%, p < 0.01). After ERAS implementation, 52% of patients were managed without the use of schedule 2 narcotics (0% pre-ERAS, p < 0.01) and 33% received no narcotics of any kind (0% pre-ERAS, p < 0.01). CONCLUSION: Implementation of an ERAS protocol for laparoscopic sleeve gastrectomy is associated with a dramatic reduction in the use of narcotics in the postoperative period. This has implementation for the usage of narcotics for laparoscopic surgery and potential elimination of narcotics for certain patients and procedures.

Effect of prenatal tramadol on postnatal cerebellar development: Role of oxidative stress.

BACKGROUND AND AIM: The adverse neurological effects of tramadol have recently raised attention. The literature pertaining to studying postnatal cerebellar changes induced by prenatal tramadol is very scanty, thus the current study has been designed to improve understanding of the cerebellar oxidative stress-related alterations associated with tramadol administration during pregnancy in this critical period of neuronal differentiation and synaptic development, thereby highlighting the importance of controlling prenatal prescription of opioids and optimizing care for opioid-dependent pregnant women and their infants. MATERIAL AND METHODS: Twenty pregnant female rats of Sprague Dawley strains were used in the study. Their offspring were divided into two groups: group I (control group) offspring of mothers given saline; group II offspring of mothers given tramadol from the 10th day (D10) of gestation till D21. The pups were sacrificed on the 7th, 14th and 21st postnatal days. Cerebellar specimens were processed for histomorphometric, immunohistochemical and electron microscopic assessment and were evaluated for various oxidative stress parameters. RESULTS: Tramadol administration during pregnancy caused profound structural abnormalities on the post-natal cerebellar cortex and was associated with oxidative stress evidenced by elevation of lipid peroxidation products and inhibition of antioxidant enzyme activities.

Modulatory role of the intra-accumbal CB1 receptor in protein level of the c-fos and pCREB/CREB ratio in the nucleus accumbens and ventral tegmental area in extinction and morphine seeking in the rats.

Brain reward and motivation circuit begin from the ventral tegmental area (VTA) that its dopaminergic terminals project to various regions of the brain including the nucleus accumbens (NAc). This reward circuit is influenced by drugs of abuse such as morphine and cannabinoid. The present study tried to investigate the role of the intra-accumbal CB1 receptor in the c-fos level and pCREB/CREB ratio in the NAc and the VTA during reinstatement phase of morphine-induced conditioned place preference (CPP) by western blotting. The present data reveals that intra-accumbal administration of CB1 agonist, WIN55,212-2 (0.5, 1 and 2 mM/0.5 µl DMSO) before/during extinction period of morphine-induced CPP, significantly decreased the NAc and the VTA c-fos protein level in the reinstatement phase; whereas the pre-reinstatement administration of the CB1 agonist, increased the c-fos protein level. Intra-accumbal administration of the CB1 agonist during the extinction period of morphine-induced CPP reduced the pCREB/CREB ratio in the NAc. Also, the present data show that intra-accumbal administration of CB1 antagonist, AM251 (15, 45 and 90 µM/0.5 µl DMSO) during/after extinction period of morphine-induced CPP affects the NAc and the VTA c-fos protein level in the reinstatement phase. Also, intra-NAc microinjection of AM251 during the extinction period reduced pCREB/CREB ratio in these regions. In conclusion, the results presented here provide compelling evidence of the modulation and involvement of the c-fos and the CREB molecules in the cannabinoid-opioid interaction of the brain reward system in the CPP paradigm.

Estrogen Regulation of GRK2 Inactivates Kappa Opioid Receptor Signaling Mediating Analgesia, But Not Aversion.

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gßγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither Grk3 nor Pdyn gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gßγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gßγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gßγ by GRK2, blunts G protein-mediated signals.SIGNIFICANCE STATEMENT Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses.

Toots, tastes and tester shots: user accounts of drug sampling methods for gauging heroin potency.

BACKGROUND: Internationally, overdose is the primary cause of death among people injecting drugs. However, since 2001, heroin-related overdose deaths in the United States (US) have risen sixfold, paralleled by a rise in the death rate attributed to synthetic opioids, particularly the fentanyls. This paper considers the adaptations some US heroin injectors are making to protect themselves from these risks. METHODS: Between 2015 and 2016, a team of ethnographers collected data through semi-structured interviews and observation captured in field notes and video recording of heroin preparation/consumption. Ninety-one current heroin injectors were interviewed (Baltimore, n = 22; Chicago, n = 24; Massachusetts and New Hampshire, n = 36; San Francisco, n = 9). Experience injecting heroin ranged from < 1-47 years. Eight participants, who were exclusively heroin snorters, were also interviewed. Data were analyzed thematically. RESULTS: Across the study sites, multiple methods of sampling "heroin" were identified, sometimes used in combination, ranging from non-injecting routes (snorting, smoking or tasting a small amount prior to injection) to injecting a partial dose and waiting. Partial injection took different forms: a "slow shot" where the user injected a portion of the solution in the syringe, keeping the needle in the injection site, and continuing or withdrawing the syringe or a "tester shot" where the solution was divided into separate injections. Other techniques included getting feedback from others using heroin of the same batch or observing those with higher tolerance injecting heroin from the same batch before judging how much to inject themselves. Although a minority of those interviewed described using these drug sampling techniques, there is clearly receptivity among some users to protecting themselves by using a variety of methods. CONCLUSIONS: The use of drug sampling as a means of preventing an overdose from injection drug use reduces the quantity absorbed at any one time allowing users to monitor drug strength and titrate their dose accordingly. Given the highly unpredictable potency of the drugs currently being sold as heroin in the US, universal precautions should be adopted more widely. Further research is needed into facilitators and barriers to the uptake of these drug sampling methods.

Resting-state Abnormalities in Heroin-dependent Individuals.

Drug addiction is a major health problem worldwide. Recent neuroimaging studies have shed light into the underlying mechanisms of drug addiction as well as its consequences to the human brain. The most vulnerable, to heroin addiction, brain regions have been reported to be specific prefrontal, parietal, occipital, and temporal regions, as well as, some subcortical regions. The brain regions involved are usually linked with reward, motivation/drive, memory/learning, inhibition as well as emotional control and seem to form circuits that interact with each other. So, along with neuroimaging studies, recent advances in resting-state dynamics might allow further assessments upon the multilayer complexity of addiction. In the current manuscript, we comprehensively review and discuss existing resting-state neuroimaging findings classified into three overlapping and interconnected groups: functional connectivity alterations, structural deficits and abnormal topological properties. Moreover, behavioral traits of heroin-addicted individuals as well as the limitations of the currently available studies are also reviewed. Finally, in need of a contemporary therapy a multimodal therapeutic approach is suggested using classical treatment practices along with current neurotechonologies, such as neurofeedback and goal-oriented video-games.

Kappa opioid receptors regulate hippocampal synaptic homeostasis and epileptogenesis.

OBJECTIVE: Homeostatic synaptic plasticity (HSP) serves as a gain control mechanism at central nervous system (CNS) synapses, including those between the dentate gyrus (DG) and CA3. Improper circuit control of DG-CA3 synapses is hypothesized to underlie epileptogenesis. Here, we sought to (1) identify compounds that preferentially modulate DG-CA3 synapses in primary neuronal culture and (2) determine if these compounds would delay or prevent epileptogenesis in vivo. METHODS: We previously developed and validated an in vitro assay to visualize the behavior of DG-CA3 synapses and predict functional changes. We used this "synapse-on-chip" assay (quantification of synapse size, number, and type using immunocytochemical markers) to dissect the mechanisms of HSP at DG-CA3 synapses. Using chemogenetic constructs and pharmacological agents we determined the signaling cascades necessary for gain control at DG-CA3 synapses. Finally, we tested the implicated cascades (using kappa opioid receptor (OR) agonists and antagonists) in two models of epileptogenesis: electrical amygdala kindling in the mouse and chemical (pentylenetetrazole) kindling in the rat. RESULTS: In vitro, synapses between DG mossy fibers (MFs) and CA3 neurons are the primary homeostatic responders during sustained periods of activity change. Kappa OR signaling is both necessary and sufficient for the homeostatic elaboration of DG-CA3 synapses, induced by presynaptic DG activity levels. Blocking kappa OR signaling in vivo attenuates the development of seizures in both mouse and rat models of epilepsy. SIGNIFICANCE: This study elucidates mechanisms by which synapses between DG granule cells and CA3 pyramidal neurons undergo activity-dependent homeostatic compensation, via OR signaling in vitro. Modulation of kappa OR signaling in vivo alters seizure progression, suggesting that breakdown of homeostatic closed-loop control at DG-CA3 synapses contributes to seizures, and that targeting endogenous homeostatic mechanisms at DG-CA3 synapses may prove useful in combating epileptogenesis.