Effect Of Chronic Morphine Treatment Research Articles

No significant change of N6 -methyladenosine modification landscape in mouse brain after morphine exposure.

N6 -methyladenosine (m6 A) plays a crucial role in regulating neuroplasticity and different brain functions at the posttranscriptional level. However, it remains unknown whether m6 A modification is involved in acute and chronic morphine exposure. In this study, we conducted a direct comparison of m6 A levels and mRNA expression of m6 A-associated factors between morphine-treated and nontreated C57BL/6 wild-type mice. We established animal models of both acute and chronic morphine treatment and confirmed the rewarding effects of chronic morphine treatment using the conditioned place preference (CPP) assay. The activation status of different brain regions in response to morphine was assessed by c-fos staining. To assess overall m6 A modification levels, we employed the m6 A dot blot assay, while mRNA levels of m6 A-associated proteins were measured using a quantitative polymerase chain reaction (qPCR) assay. These analyses were performed to investigate whether and how m6 A modification and m6 A-associated protein expression will change following morphine exposure. The overall m6 A methylation and mRNA levels of m6 A-associated proteins were not significantly altered in brain regions that were either activated or not activated during acute morphine stimulation. Similarly, the overall m6 A modification and mRNA levels of m6 A-associated proteins remained unaffected in several key brain regions associated with reward following chronic morphine exposure. This study showed that the overall m6 A modification level and mRNA expression levels of m6 A-associated factors were not affected after acute and chronic morphine exposure in different brain regions, indicating m6 A modification may not be involved in brain response to morphine exposure.

Chronic Morphine Induces Adaptations in Opioid Receptor Signaling in a Thalamostriatal Circuit That Are Location Dependent, Sex Specific, and Regulated by μ-Opioid Receptor Phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) via activity at μ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in the DMS. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation) in male, but not female, mice. At MThal cell bodies, chronic morphine treatment decreased subsequent morphine activation of potassium conductance (morphine tolerance) in both male and female mice. In knock-in mice expressing phosphorylation-deficient MORs, chronic morphine treatment resulted in tolerance to, rather than facilitation of, subsequent morphine signaling at MThal-DMS terminals, suggesting phosphorylation deficiency unmasks adaptations that counter the facilitation observed at presynaptic terminals in wild-type mice. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry, and sex.

Repeated Endomorphin Analogue MEL-0614 Reduces Tolerance and Improves Chronic Postoperative Pain without Modulating the P2X7R Signaling Pathway.

The clinical treatment of chronic postoperative pain (CPSP) remains challenging. The side effects of chronic morphine treatment limit its clinical application. MEL-0614, a novel endomorphin analogue that is highly selective and agonistic for μ opioid receptor (MOR), produces a more powerful analgesic effect than that of morphine. In this study, we explored the difference in antinociceptive tolerance and related mechanisms between MEL-0614 and morphine in CPSP induced in a skin/muscle incision and retraction (SMIR) mice model. We found that acute administration of MEL-0614 (1, 3, 5, and 10 nmol, i.t.) produced a dose-dependent analgesic effect that was superior to that of morphine in the SMIR mice model. Long-term MEL-0614 treatment (10 nmol, i.t.) did not induce tolerance compared with morphine. Notably, tolerance induced by morphine could be greatly prevented and/or inhibited via cross-administration or coadministration between MEL-0614 and morphine. In addition, MEL-0614 accelerated the recovery of postoperative pain, whereas morphine aggravated postoperative pain and prolonged its recovery time regardless of preoperative or postoperative treatment. In addition, MEL-0614 did not activate microglia and the P2X7R signaling pathway and showed reduced expression iba1 and P2X7R compared with that observed after morphine administration. Release of inflammatory factors was induced by continued administration of morphine during SMIR surgery, but MEL-0614 did not promote the activation of inflammatory factors. Our results showed that MEL-0614 has superior analgesic effects in CPSP and leads to tolerance to a lesser degree than morphine. Further, MEL-0614 may be used as a promising treatment option for the long-term treatment in CPSP.

Sex differences in MAP kinase activation in the periaqueductal gray after morphine treatment

Morphine is a potent opioid analgesic, but its long term use can lead to negative side effects, including tolerance, which is a decrease in the effectiveness of the opioid. An area of active interest is looking into the molecular effects of chronic morphine treatment in the Periaqueductal gray (PAG), a brain region that controls descending pain modulation. One such molecular target within the PAG is extracellular‐signal regulated kinase 1/2 (ERK). Previous studies have shown that pharmacological inhibition of ERK enhanced morphine tolerance, indicating that ERK activity is associated with better responsiveness to morphine. The PAG is known to contain a heterogenous population of neurons including GABA and glutamate subtypes. However, which neurons ERK is activated in within the PAG following morphine tolerance is unknown. Further, there are known differences in PAG activity between male and female mice. These sex‐differences have not been well studied after morphine tolerance using acute pain tests. The purpose of this research is to investigate differences in ERK activation following morphine tolerance in male and female mice. We treated wild‐type male and female mice with morphine (10 mg/kg, i.p.) or saline for 5 days to induce morphine tolerance, following which both behavior and protein immunofluorescence were assessed. We observe sex‐specific differences in ERK activation levels and morphine antinociceptive tolerance in mice. We also assessed co‐localization of ERK with GABA and glutamate neurons after morphine tolerance. The study will help us understand the cell‐type specificity of kinase activation following morphine tolerance. Further this will give us more information about the nature of neurons that are contributing to sex‐differences in opioid functions within the PAG.

Morphine-Induced Dendritic Spine Remodeling in Rat Nucleus Accumbens Is Corticosterone Dependent.

BackgroundChronic morphine treatments produce important morphological changes in multiple brain areas including the nucleus accumbens.MethodsIn this study, we have investigated the effect of chronic morphine treatment at a relatively low dose on the morphology of medium spiny neurons in the core and shell of the nucleus accumbens in rats 1 day after the last injection of a chronic morphine treatment (5 mg/kg once per day for 14 days). Medium spiny neurons were labeled with 1,1’ dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate crystal and analyzed by confocal laser-scanning microscope.ResultsOur results show an increase of thin spines and a decrease of stubby spines specifically in the shell of morphine-treated rats compared with control. Since morphine-treated rats also presented an elevation of corticosterone level in plasma, we explored whether spine alterations induced by morphine treatment in the nucleus accumbens could be affected by the depletion of the hormone. Thus, bilaterally adrenalectomized rats were treated with morphine in the same conditions. No more alteration in stubby spines in the shell was detected in morphine-treated rats with a depletion of corticosterone, while a significant increase was observed in mushroom spines in the shell and stubby spines in the core. Regarding the thin spines, the increase observed with morphine compared with saline was lower in adrenalectomized rats than in nonadrenalectomized animals.ConclusionThese results indicate that dendritic spine remodeling in nucleus accumbens following chronic morphine treatment at relatively low doses is dependent on corticosterone levels.

Effect of Chronic Opioid Therapy on Pain and Survival in a Mouse Model of Sickle Cell Disease

Pain is one of the major comorbidities of sickle cell disease (SCD) requiring chronic opioid therapy (COT). However, COT has been associated with reduced survival and opioid-induced hyperalgesia (OIH), which can exacerbate the chronic pain and increased mortality already inherent to SCD. It is challenging to examine pain, opioid use and survival in populations with SCD due to significant heterogeneity in multiple clinicopathologic factors. To determine the effect of COT we used transgenic HbSS BERK sickle mice, which show pain characteristics similar to those observed in patients with SCD, including sensitivity to thermal and mechanical stimuli, increased opioid requirement, and higher levels of circulating substance P and tryptase (Tran et al., Blood 2017). Additionally, similar to female patients with SCD, female BERK sickle mice show more pain than males. Therefore, we performed a randomized double-blind placebo-controlled trial to examine the effect of COT on pain and survival in female homozygous HbSS BERK (sickle) and HbAA BERK (control) mice, expressing >99% human sickle hemoglobin and normal human hemoglobin A, respectively. Mice were injected subcutaneously each day with either morphine sulfate at a starting dose of 20 mg/kg, which was increased to 25 mg/kg, 30 mg/kg, 35 mg/kg, and 40 mg/kg after weeks 12, 18, 28, 30, and 38 respectively or placebo (equal volume of saline) until the end of survival. Dose of morphine was selected based on the effective analgesic dose for these mice (Kohli et al., Blood 2010). Mechanical-, thermal- (heat and cold), and musculoskeletal/deep hyperalgesia were analyzed in all mice biweekly before and after drug treatments. We used Kaplan-Meier and log-rank test with Sidak correction for survival; 2-way repeated measures analysis of variance with Bonferroni's correction to compare hyperalgesia between time points among control and sickle mice and Cox proportional-hazards regression analysis for the association of hyperalgesia with survival.We observed significantly decreased survival of saline-treated sickle mice compared to saline-treated control mice (P = 0.0009). Compared to saline, morphine treatment led to a significant decrease in survival in control mice (P = 0.035) but not in sickle mice (P > 0.05). Furthermore, we did not observe an association between hyperalgesia and survival in either control or sickle mice. However, we discerned a significant increase in mechanical, cold, and heat hyperalgesia in control mice after 4 weeks of morphine treatment (P < 0.02, compared to day '0') which continued to increase up to 12 weeks (P <0.05 compared to week 4). Similarly, in sickle mice we observed an increase in mechanical hyperalgesia after 4 weeks (P <0.02, compared to day '0') of morphine treatment which continued to increase up to 12 weeks (P < 0.0001). These data suggest COT leads to OIH in both control and sickle mice. It is noteworthy that morphine treatment continued to show an analgesic effect over the course of 12 weeks in spite of an increase in hyperalgesia in both groups of mice. Thus, mice did not develop tolerance to morphine analgesia. Since mice were treated under uniform conditions, the effect of chronic morphine treatment could be observed without any confounding factors. Our findings in control mice recapitulate the clinical observations that COT is associated with reduced lifespan in non-sickle patients. COT lead to OIH in sickle mice but provided analgesia without causing tolerance or reducing survival. OIH may contribute may exacerbate a vicious cycle of chronic pain and opioid use in SCD. Differences exist between humans and mice including morphine metabolism between the two but due to several similarities of HbSS BERK sickle mice with clinical pain characteristics and biology, these observations have a significant translational potential following clinical trials for optimizing the treatment of pain in SCD. These data highlight the critical need to develop analgesic strategies devoid of OIH. DisclosuresGupta:Tau tona: Consultancy; Novartis: Honoraria.

Morphine-potentiated cognitive deficits correlate to suppressed hippocampal iNOS RNA expression and an absent type 1 interferon response in LP-BM5 murine AIDS

Opioid use accelerates neurocognitive impairment in HIV/AIDS patients. We assessed the effect of chronic morphine treatment and LP-BM5/murine AIDS (MAIDS) infection on cognition, cytokine production, and type 1 interferon (IFN) expression in the murine CNS. Morphine treatment decreased expression of pro-inflammatory factors (CCL5, iNOS) and reduced cognitive performance in LP-BM5-infected mice, correlating to increased hippocampal viral load and a blunted type 1 IFN response. In the striatum, morphine reduced viral load while increasing IFN-α RNA expression. Our results suggest that differentially regulated type 1 IFN responses may contribute to distinct regional outcomes in the hippocampus and striatum in LP-BM5/MAIDS.

(249) - Role of nerve growth factor/tyrosine kinase receptor a signaling in the plasma of morphine tolerant rat

Morphine is extensively used as an analgesic in pain management. However, repeated administration of morphine produces tolerance. In the central nervous system, nerve growth factor (NGF) and its receptor, tyrosine kinase receptor A (TrkA), are involved in pain, analgesia, and tolerance mechanisms. Additionally, multiple clinical and animal studies have shown changes in circulating NGF in various neurological disorders. In the present study, we investigated the effects of chronic morphine treatment on the NGF-TrkA pathway in morphine-induced tolerant rats. Animals were treated with morphine with or without concomitant administration of the tyrosine kinase receptor A antagonist K252a. The morphine (10 mg/kg; subcutaneous) or K252a (80 µg/kg; intraperitoneal) treatment regime was 2x/day for five days. On the sixth day, a single injection of morphine/K252a was administered. The development of tolerance was assessed by changes in paw withdrawal latency (PWL) to noxious thermal stimuli (Thermal test). The change in plasma NGF levels was measured by ELISA. PWL progressively decreased in rats that received repeated morphine treatments indicating development of antinociceptive and morphine tolerance. Morphine tolerant rats showed two fold increases in the plasma NGF level compared to saline-treated control rats. Co-administration of morphine and K252a increased PWL and also reduced the plasma NGF levels. These initial findings suggest that plasma NGF-TrkA signaling contributes to the development of opioid tolerance. This research was supported by Congressionally Directed Medical Research Programs- Applied Pain Research grant (MR157005C).

Chronic administration of morphine using mini-osmotic pumps affects spatial memory in the male rat

The use of opioid analgesics to treat non-cancer pain has increased over the years. Many chronic pain patients suffer from numerous adverse effects, such as reduced quality of life, development of dependence, and cognitive impairments. Cognitive processes are regulated by several systems, one of which involves growth hormone (GH) and its secondary mediator insulin-like growth factor-1 (IGF-1), but also glutamatergic transmission, including receptors such as the N-methyl-d-aspartate (NMDA)-receptor complex. In the laboratory, repeated injections are commonly used to establish animal models of long-term or chronic drug exposure. However, in the present study, we aimed to mimic a more human dose regimen using constant drug delivery provided by mini-osmotic pumps implanted subcutaneously in male Sprague Dawley rats. After developing opioid tolerance the cognitive function of rats was studied. Spatial learning and memory capabilities were evaluated using the rat Morris water maze (MWM). Moreover, gene expression related to the GH/IGF-1-axis and the NMDA-receptor system was analyzed using quantitative PCR (qPCR) and plasma levels of IGF-1 were assessed using the ELISA technique. Our results demonstrate that rats exposed to morphine for 27 days display memory impairments in the MWM probe trial. However, the behavioral effects of chronic morphine treatment were not accompanied by any significant differences in terms of mRNA expression or IGF-1 plasma concentration. The animal model used in this study provides a simple and suitable way to investigate the behavioral and neurochemical effects of chronic opioid treatment similar to the exposure seen in human pain patients.

Chronic Morphine Reduces Surface Expression of δ-Opioid Receptors in Subregions of Rostral Striatum.

The delta opioid receptor (DOPr), whilst not the primary target of clinically used opioids, is involved in development of opioid tolerance and addiction. There is growing evidence that DOPr trafficking is involved in drug addiction, e.g., a range of studies have shown increased plasma membrane DOPr insertion during chronic treatment with opioids. The present study used a transgenic mouse model in which the C-terminal of the DOPr is tagged with enhanced-green fluorescence protein to examine the effects of chronic morphine treatment on surface membrane expression in striatal cholinergic interneurons that are implicated in motivated learning following both chronic morphine and morphine sensitization treatment schedules in male mice. A sex difference was noted throughout the anterior striatum, which was most prominent in the nucleus accumbens core region. Incontrast with previous studies in other neurons, chronic exposure to a high dose of morphine for 6days had no effect, or slightly decreased (anterior dorsolateral striatum) surface DOPr expression. A morphine sensitization schedule produced similar results with a significant decrease in surface DOPr expression in nucleus accumbens shell. These results suggest that chronic morphine and morphine sensitisation treatment may have effects on instrumental reward-seeking behaviours and learning processes related to drug addiction, via effects on striatal DOPr function.

Effects of Chronic Morphine Treatment on an Odor Conditioning Paradigm, Locomotor Activity and Sucrose Responsiveness in Honeybees (Apis mellifera)

Honeybee is a widely used insect model for learning and memory research. Recently, it has become a potentially good subject for evaluating the effects of addictive drugs on the nervous systems. Our previous study has found that acute morphine injection affected associative memory and locomotor activity in honeybees. In the current study, the effect of chronic morphine treatment and its cessation in honeybees were assessed. The results demonstrated that 1) chronic morphine (0.01, 0.1 and 1 mg/ml) treatment for 7 days severely diminished associative memory in honeybees; 2) 1 mg/ml morphine consumption for 5, 7 and 10 but not 3 days impaired the olfactory memory; 3) Bees withdrawn from morphine for 1 day but not 3 days showed amnesia in the PER conditioning. We also found that bees displayed hyperactivity and tolerance in response to chronic morphine administration. In addition, morphine dose-dependently altered the sucrose responsiveness of bees. The data indicated that chronic morphine has sensory-motor effects and may impair learning and/or memory in honeybees, which were comparative to that in vertebrates.

544 Uroguanylin-GUCY2C Brain Gut Axis Regulating Feeding Behavior Is Mediated by Nodose Ganglia HCN2 Ion Channel

Opiates differ in their ability to trigger μOR endocytosis and to promote physiological tolerance, which involve changes at the receptor levels and downstream of the receptor. The aims of this study were to test whether agonists with different internalizing abilities activate MAPK, a μOR downstream signaling, in enteric neurons, and the effect of chronic morphine treatment on opioid-induced activation of MAPK. Cultured rat myenteric neurons were incubated with 1 μM DAMGO, a potent μOR-internalizing agonist or 10 μM morphine, a poor μOR-internalizing agonist for 0'-30' at 37oC. MAPK or extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was determined byWestern Blot using membranes from cell lysates and the phospho-p44/p42 MAPK and total ERK1/2 antibodies. Specificity controls included pre-incubation with the opioid receptor antagonist, naloxone (10 μM), tetrodotoxin (TTX; 10μM) to block neurotransmitter release or the MAPK inhibitor, U0126 (10 μM). To determine whether opioid-induced MAPK requires receptor internalization and the role of the intracellular trafficking protein, dynamin, myenteric neurons were treated with hypertonic sucrose solution (0.5 M) to block endocytosis, with the dynamin inhibitor, dynasore (10μM) prior to opioid stimulation, or transfected with a wild-type dynamin or a mutated-dynamin using lentiviral vectors. There was a significant increase in MAPK phosphorylation in neurons exposed to DAMGO at 5' and 10', which returned to control levels at 20', while morphine did not induce MAPK activation. DAMGO-induced MAPK phosphorylation was blocked by naloxone and MAPK inhibitor U0126, and by sucrose, dynasore and mutated-dynamin transfection. TTX did not prevent MAPK phosphorylation. To determine the effect of chronic μOR activation on opioid-induced MAPK phosphorylation, cells were treated with 10 μM morphine for 4 days before exposure to DAMGO or morphine. Both DAMGO and morphine acute stimulation induced MAPK activation in enteric neurons chronically treated with morphine, with a peak at 5' and 10' returning to control levels at 20' as in normal cells. Stimulation with DAMGObut not morphine induces μOR internalization in Naive myenteric neurons, whereas both DAMGO and morphine stimulation trigger μOR internalization in neurons chronically treated with morphine. This study shows that opioid stimulation induces a rapid and transient activation of MAPK/ERK in enteric neurons, which is ligand dependent and mediated by direct interaction of the ligand with μOR. Opioid stimulation of MAPK/ERK pathway in enteric neurons requires μOR internalization and is dynamin-dependent. This suggests that receptor endocytosis regulates MAPK, which in turn could trigger feedback signaling affecting cellular response and adaptations, mechanisms underlying the development of chronic opiate adverse effects.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Sex differences in the Nociceptin/Orphanin FQ system in rat spinal cord following chronic morphine treatment

Nociceptin/Orphanin FQ (N/OFQ) appears to contribute to the development of morphine tolerance, as blockade of its actions will block or reverse the process. To better understand the contribution of N/OFQ to the development of morphine tolerance, this study examined the effect of chronic morphine treatment on levels of N/OFQ and levels and activity of the N/OFQ peptide (NOP) receptor in spinal cord (SC) from male and female rats. Both male and female Wistar rats showed less responsiveness to morphine after subcutaneous injection of escalating doses of morphine (10, 20, 40, 60 and 80 mg/kg, respectively) twice daily for five consecutive days. Male rats were more tolerant to the antinociceptive actions of morphine than females. The N/OFQ content of SC extracts was higher in females than in males, regardless of treatment; following chronic morphine treatment the difference in N/OFQ levels between males and females was more pronounced. N/OFQ content in cerebrospinal fluid (CSF) was reduced 40% in male and 16% in female rats with chronic morphine exposure, but increased in periaqueductal grey of both sexes. Chronic morphine treatment increased NOP receptor levels 173% in males and 137% in females, while decreasing affinity in both. Chronic morphine increased the efficacy of N/OFQ-stimulated [35S]GTPγS binding to SC membranes from male rats, consistent with increased receptor levels. Taken together, these findings demonstrate sex differences in N/OFQ–NOP receptor expression and NOP receptor activity following chronic morphine treatment. They also suggest interplay between endogenous N/OFQ and chronic morphine treatment that results in nociceptive modulation.

The Effect of Chronic Morphine Treatment on the Abundance of Cell Signaling Proteins in the Guinea Pig Longitudinal Muscle‐Myenteric Plexus

Chronic exposure to morphine produces heterologous tolerance in the longitudinal muscle/myenteric plexus (LM/MP) of the guinea pig. A decrease in the α3 subunit of the sodium pump was suggested as one mechanism since it develops over a time course similar to the reduced function. However, a large number of cell signaling proteins have also been proposed as contributors to the adaptation process. We examined the abundance of several different proteins using quantitative western blot analysis in homogenates of LM/MP after chronic morphine exposure. We found no change in the abundance of the mu opioid receptor protein either 4 or 7 days after pellet implantation when the loss of function occurs and becomes maximal. We also found no change in the abundance of the α1 subunit isoform of the sodium pump, PKCε, beta actin or GAPDH at any time period from 1–14 days after exposure. In contrast, we observed an increase in the abundance of calcineurin, GRK2, and Giα2 and a decrease in the abundance of PKCγ and the alpha3 subunit isoform at 7 days after implantation. The lack of correlation of changes in protein abundance with functional changes suggests that the heterologous tolerance that develops after chronic morphine treatment may involve multiple cellular mechanisms including the modulation of cell excitability and normal physiology which result from changes in the abundance of proteins in several signaling pathways.

Chronic Morphine Treatment Switches the Effect of Dopamine on Excitatory Synaptic Transmission from Inhibition to Excitation in Pyramidal Cells of the Basolateral Amygdala

Dopaminergic signaling in the basolateral amygdala (BLA) is important for drug-stimulus learning that triggers relapse to drug-seeking behavior. However, little is known about adaptive changes in this signaling pathway upon chronic morphine treatment. In this paper, we observed the influence of chronic morphine treatment on the effect of dopamine (DA) on the excitatory transmission in the pyramidal cells of BLA in slices with the whole-cell patch-clamp method. We also studied its mechanism and significance with pharmacological approaches combined with biochemical and behavioral techniques. The results showed that chronic morphine exposure switched the effect of DA on the excitatory synaptic transmission from inhibition to excitation; the chronic morphine-induced switching action on the effect of DA was due to its influence on D1 receptors; the site of the effect of chronic morphine treatment on D1 receptors was at presynaptic locus; chronic morphine treatment induced a significant increase in the amount of D1 receptor expression in the synaptosomes and synaptosomal membrane fraction from BLA; the enhancement of presynaptic glutamate release by D1 receptor agonist upon chronic morphine treatment was dependent on the activation of cAMP-dependent protein kinase; and the intra-BLA injection of D1 receptor antagonist canceled the conditioned place aversion (CPA) in morphine-dependent rats. In conclusion, chronic morphine treatment switches the effect of DA on the excitatory synaptic transmission from inhibition to excitation by the presynaptic D1 receptor amount increase-mediated glutamate release in the pyramidal cells of BLA and the blockade of D1 receptors in BLA cancels CPA in morphine-dependent rats.

The Role of β-Arrestin2 in the Mechanism of Morphine Tolerance in the Mouse and Guinea Pig Gastrointestinal Tract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Effect of chronic morphine treatment on tactile learning in rat

In our previous study, we reported that chronic morphine exposure changes neuronal response properties in rat somatosensory cortex. In this study, we investigated the effect of chronic morphine treatment on tactile learning behavior in rats. Morphine sulfate was dissolved in tap water, and was administered for 21 days. Tactile learning was assessed using the novel object recognition test (NORT) in a dark room. The chronic morphine treated group exhibited a learning impairment; the discrimination ratio was significantly lower as compared to the control group. These findings suggest that chronic exposure to morphine impaired the tactile learning in rats as the discrimination ratio was decreased following morphine administration.

Effects of chronic morphine treatment on protein expression in rat dorsal root ganglia

The initial aim of this study was to identify protein changes in the dorsal root ganglia (DRG) associated with long-term morphine treatment. We carried out a differential proteomics analysis on samples from the DRG of control and morphine-dependent rats (5–40 mg/kg, subcutaneously, twice daily for 28 days) after 4 days of morphine withdrawal. Proteins showing a statistically significant variation between the two groups were selected for identification by mass spectrometric analysis. Twelve proteins were unambiguously identified, with the majority being enzymes involved in energy metabolism and protein degradation, signaling and cytoskeletal proteins. Aldolase C and proteasome subunit α type 3 (PRC8 or α7) were further examined by Western blot analysis in the DRG, prefrontal cortex, nucleus accumbens, striatum, hippocampus, ventral tegmental area and locus coeruleus. In addition, expression of PRC8 in the nucleus accumbens tissue after spontaneous or naloxone-precipitated withdrawal was determined using immunohistochemistical staining. Our results indicate that the expression levels of aldolase C and PRC8 proteins were altered in a time- and region-specific manner, and suggest that they could be implicated in the molecular changes underlying adaptations of neurons observed with chronic morphine treatment.

Effects of aquaporin4 deficiency on opioid receptors characteristics in naive and chronic morphine-treated mice

Our previous study indicated that aquaporin4 (AQP4) deficiency potentiated morphine analgesia, but attenuated tolerance and physical dependence induced by chronic exposure to morphine. However, the mechanisms remained to be explored. In the present study, effects of AQP4 deficiency on opioid receptor characteristics were investigated by [ 3H]-diprenorphine binding assays. In basal condition, the K d values of opioid receptors increased from 0.27 ± 0.03 nM in wild-type mice to 0.44 ± 0.04 nM in AQP4 deficient mice. Meanwhile, the density ( B max values) of opioid receptors increased from 0.40 ± 0.04 pmol/mg protein in wild-type mice to 0.66 ± 0.04 pmol/mg protein in AQP4 deficient mice. After chronic morphine treatment, the affinity of opioid receptors decreased in wild-type mice, in which the K d value increased from 0.27 ± 0.03 nM to 0.40 ± 0.04 nM, while no change in the density of opioid receptors was observed. In AQP4 knockout mice, the effects of chronic morphine treatment on opioid receptors were similar to that in wild-type mice, in which the K d values increased from 0.44 ± 0.04 nM to 0.64 ± 0.08 nM, whereas the density had no significant change. Taken together, at the first time, we found that AQP4 deficiency decreased the affinity and increased the density of opioid receptors. Additionally, AQP4 deficiency did not affect chronic morphine-induced alterations of opioid receptor characteristics.