Neuron-Microglia Communication Delivered CRFR1 Phosphorylation during Long-Term Morphine Treatment
Corresponding author: Dr. Hui Zhao, Department of Integrative Medicine and Neurobiology, Shanghai Medical College, Fudan University, 138#Yixueyuan Rd., Tel: 86-21-54237611; Fax: 86-21-54237023;
Chronic use of morphine is thought to induce neuronal adaptations and synaptic plasticity in specific brain regions, changes ultimately contribute to the addictive phenotype [1-4]. It has been recognized that development of morphine dependence and tolerance was associated with neuronal opioid receptor-dependent cAMP signal transduction networks[ 5-7], for example, the initially decreases in cAMP levels by MOR activation could be restored during continued exposure to morphine (tolerance), and even are increased further (superactivation) upon opiate receptor blockade or opiate withdrawal [8-16].
In principal, morphine mediated AC superactivation was dependent on the signaling compartmentalization within lipid rafts, c-Src could facilitate temporal and spatial translocation of calreticulin (CRT), a ER chaperone, from ER lumen to the lipid rafts, wherein assembling cAMP signaling via adhesion or contact formation[17-19]. Recently, it was greatly gathered an association between neuroinflammation polymorphisms and risk of morphine dependence and tolerance [20-21]. Interoceptive cues provided by the up-regulation of microglia activation derived proinflammatory mediators might be sufficient stimuli to morphine withdrawal severity [22-24]. Mounting evidence indicated that surveillance of the CNS occurred through specialized neuron-glia communication, adhesion molecules including CD44 could directly allow “eat-me” signals to prevail, forming a neuroinflammation associated molecular patterns [25-34]. Then, it was presumed that microglia primed microenvironment might promote morphine mediated AC superactivation.
At present, it is identified that CD44 is dependent on the extracellular structure that can be linked to Src kinase, which was thought to coordinate the stress response via regulating hypothalamic-pituitary-adrenal axis [35-36]. Since the negative affective states of withdrawal mostly involve recruitment of brain stress neurocircuitry, for example, CRF1R subtype was reported to be related with behavioral and autonomic activation that occurs following morphine withdrawal [37-41]. However, coordination of CRFR specialization and MOR signaling during long-term morphine treatment is open question.
Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states [42-43]. As such, molecular connection of RGS4 with MOR or CRFR1 is an interesting mechanistic problem, it is possible that microglia activation establish a cellular communication, by which maintain and potentiate a signaling switch between MOR and CRFR1 during morphine dependence and tolerance.
Materials and Methods
For morphine treatment, mice were s.c. injected with escalating doses of morphine twice daily for 5 days. On day 6, mice were injected with 1 mg/kg naloxone 2h after 100 mg/kg morphine treatment. Several measures of opiate withdrawal were assessed as followed, mice were placed inside transparent cylinder (50x30cm) under a 50 lux indirect lighting, and observed for 30 min, somatic signs of jumps, wet dog shakes, paw tremor, rear and grooming were monitored and quantified.
Morris Water Maze was performed as reported . In the acquisition section, the mice were placed in the water maze (122 cm diameter) with the platform (10 cm diameter) submerged in the SW quadrant. The mice were trained for four trails per day for 5 days with a semi-random set of start positions. AnyMaze software was used to track the mice. In the extinction section, mice were placed in the NE quadrant of the water maze without the platform for 60s. The amount of time the mice spent in each quadrant searching for the platform was recorded. Probe tests were performed every 4 days afterward until the mice spent an equal amount of time in each quadrant. Additionally, hot-plate test was performed, the cutoff time was set at 10s, antinociceptive response is expressed as the mean ± S.E.M. of percentage of the maximum possible effect (%MPE).
For treatments, mice were positioned in a Kopf stereotaxic apparatus and burr holes drilled into the skull and a 5μl Hamilton syringe fitted with a 33-gauge needle was lowered into prefrontal cortex for drug delivery. Adenovirus particles (5×109 plaque-forming units, PFU) dissolved in sterilized PBS was injected over 10s via the needle at a volume of 2μl. All experimental procedures were approved by Ethics Committee of Fudan University, efforts were made to minimize the numbers of animal used and their suffering. Animals were conducted in accordance with the relevant codes of practice for the care and use of animals for scientific purposes (National Institutes of Health, 1985).
For neuron cultures, mice fetuses were removed from pregnant rats on embryonic day 18. Cortices were dissected and collected in Hanks’ balanced salt solution. Cells were dissociated and plated at a density of 10 cells per well into 24-well tissue culture plates pretreated with 0.1% polyethylenemine. Cells were maintained in serum-free Neurobasal medium containing B27 supplement (Gibco, Rockville, MD). After 3-4 days in culture, neurons sent out long processes. By 10 days, flow cytometry showed that MAP2 immunopositive cells accounted for more than 95% of cells.
For astrocyte cultures, the dissociated cells were plated in untreated 24 well tissue culture plates. The culture medium was Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 U penicillin/ 50 μg/ml streptomycin, the adherent cells were purified after 24 h plating and cultured for another 2 weeks. For microglia culture, microglia was isolated from above the nonadherent cells, after grown for 2 weeks with the medium changed twice a week, then the cells were cultured for an additional 10 days without changing the medium to provoke nutritional deprivation, which promotes the appearance of microglia. Loosely adherent cells were recovered by gently flushing culture medium and plated at a density of 105 cells per well into 24-well tissue culture plates. After 6 days plating, the cells were treated and assayed.
In vitro Src kinase assay
Proteins were separated and immunoprecipitated with antic-Src antibody (1:200; Abcam), the resulting pellets were washed with acetone and incubated at 30°C with 5 μg of SRC substrate peptide (KVEKIGEGTYGVVYK, corresponding to amino acids 6-20 of p34cdc2; Upstate Biotechnology, Lake Placid, New York) in kinase buffer containing 5 μCi of [γ-32P]-adenosine triphosphate ([γ-32P]-ATP; PerkinElmer Life Sciences, altham, Massachusetts), 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 10 mM MnCl2, 25μM ATPase, 1 mM dithiothreitol, and 100μM Na3VO4. After 30 min, the reaction was terminated by the addition of 10μl of 40% (w/v) TCA, and samples were spotted on P81 cellulose phosphate paper (Upstate Biotech). The paper was washed three times with 1% (w/v) phosphoric acid and once with acetone. Radioactivity retained on the P81 paper was quantified by liquid scintillation counting. Blank counts (without tissue lysate) were subtracted from each result, and radioactivity (cpm) was converted to picomoles per minute (pmol/min).
Mice were deeply anesthetized with 10% chloral hydrate anesthesia (4 ml/kg, i.p.) and transcardially perfused with 4% paraformaldehyde. Brains were removed and postfixed overnight, then equilibrated at 4°C in 30% sucrose/PBS (0.01 M, NaCl 0.15 M, pH 7.4) for at least 3 d before sectioning. Brains were sectioned at 20μm thickness on a freezing microstome (Leica, SM2000R). Every sixth serially obtained section was probed against anti-CRFR1 and Alexa Fluor488 conjugated secondary antibody. Data derived from each group were analyzed by Leika Q500IW image analysis system. For statistical analysis, fluorescent density is reported as the average density of all individual animals±SEM in each experimental group (n=5). The mean density of all the microphotographs was analyzed with the aid of ImageJ analysis software.
Prefrontal cortex was dissected and homogenized in ice-cold lysis buffer containing 137mM NaCl, 10mM Tris-HCl, pH8.0, 1mM EDTA, pH 8.0, 1% NP-40, 10% glycerol, 1mM phenylmethylsulphonyl fluoride, 10mg/ml aprotinin, 1mg/ml leupeptin, and 0.5mM sodium vanadate. The tissue homogenate solutions were centrifuged at 14,000 × g for 5 min at 4°C. The supernatants were collected and used for quantification of total protein. Levels of IL-1β, iNOS, MCP-1, MIPα, MIPβ, and TNF-α were assessed using a commercially available assay kit from Promega. Color change was measured in an ELISA plate reader at 450 nm.
Nuclear extracts were prepared from tissue or cells. Briefly, 1ml of extraction buffer was added (10mM Hepes, pH7.9, 1.5mM MgCl2, 10mM KCl) together with the recommended amount of protease inhibitor cocktails. After three freezethaw cycles, cytoplasmic extracts were recovered by centrifugation at 15,000 × g for 5 min, and pellets were resuspended in buffer C (20mM Hepes, pH7.9, 1.5mM MgCl2, 420mM KCl, 0.2mM EDTA, 25% glycerol) together with the recommended amount of protease inhibitor cocktails. Following a 30min incubation at 4°C, nuclear extracts were recovered by centrifugation at 15,000 × g for 5 min.
Immunoprecipitation and Western Blotting
Prefrontal cortex was homogenized and centrifuged, the supernatants were incubated with anti–CRFR1, anti-MOR antibodies (1:200; Santa Crutz Biotechnology; Santa Cruz, CA) at 4°C overnight with slow rotation. 60μl of protein G–agarose beads (Invitrogen, Carlsbad, CA) were added and further incubated for 3h. Afterwards, the beads were washed and protein sample were eluted from with 1x SDS sample buffer. For western Blot analysis, proteins were resolved in SDSpolyacrylamide gel, and transferred to polyvinylidene difluoride membrane (GE Healthcare, Piscataway, NY). The membrane was probed in the presence of anti-c-Src or anti-CD44 (1:1000; BD Transduction Laboratories, Lexington, KY), anti-RGS4 (1:1000, Abcam), anti-AC5 antibodies (1:1000, Santa Crutz Biotechnology). Then the membrane was incubated with secondary antibody conjugated with alkaline phosphatase, protein bands were detected by ECF substrate and scanned in the Storm 860 Imaging System. The band intensities were quantified and analyzed with the ImageQuant software (GE Healthcare).
Brain tissues were homogenized in 70μl of ice cold Krebs-Henseleit (KRBS) buffer: 118.5mM NaCl, 4.7mM KCl, 1.18mM MgSO4, 2.5mM CaCl2, 1.18mM KH2PO4, 24.9mM NaHCO3, 10mM dextrose, 10μg/ml adenosine deaminase, pH7.4, 350μl of protease inhibitor cocktails. The homogenate was diluted with 350 μl of additional ice-cold buffer. This mixture was loaded into a 1ml Tuberculin syringe attached to a 13mm diameter Millipore syringe filter holder. The diluted filtrate was forced over three layers of nylon (Tetko, 100μm pore size) pre-wetted with 150μl of KRBS, and collected in a 1.5ml Eppendorf tube. The filtered particulate was then spun at 1000×g for 15min in a microfuge at 4°C. The resultant pellet, containing synaptoneurosome fraction was used for later analysis. Protein concentration was determined using the BioRad Protein Assay.
Functional [35S]GTPγS binding
Membrane preparations of cells were diluted in 50mM Tris-HCl buffer (pH7.4) to get appropriate protein content for the assays (∼10 μg of protein/sample). Membrane fractions were incubated at 30°C for 60 min in Tris-EGTA buffer (pH7.4) composed of 50mM Tris-HCl, 1mM EGTA, 3mM MgCl2, 100mM NaCl, containing 20MBq/0.05 cm3 [35S] GTPγS (0.05nM) and morphine (1μM, 5min or 4h) in the presence of excess GDP (30μM). Total binding was measured in the absence of test compounds, non-specific binding was determined in the presence of 10μM unlabeled GTPγS and subtracted fromtotal binding. [35S]GTPγS incorporation was separated by vacuum filtration through Whatman GF/B filters with cell harvester. Filters were washed three times with 5ml ice-cold buffer (pH 7.4), and the radioactivity retained on the dried filters was quantified by liquid scintillation counter. The experiment were performed in triplicates and repeated at least three times.
Recombinant adenovirus construction
Recombinant adenovirus expressing mice CRFR1 or CRFR- 386A was constructed by inserting into the adenoviral shuttle vector pDE1sp1A (Microbix Biosystems, Inc. Canada), and the insert was then switched to the adenoviral vector through LR recombination. Adenovirus was purified by CsCl2 gradients and PD-10 Sephadex chromatography. After homologous recombination with the backbone vector PJM17, plaques resulting from viral cytopathic effects were selected and expanded in 293 cells.
Intracellular cAMP Level
Approximately 4х104 neurons/well were seeded in 96-well plates 24h before the assay. After the cells were treated with 1μM morphine for the indicated time, the medium was removed and replaced with 100μl of reaction buffer (0.5mM 3-isobutyl-1-methylxanthine and 10μM forskolin in Krebs- Ringer-HEPES buffer (110mM NaCl, 25mM glucose, 55mM sucrose, 10mM HEPES, 5mM KCl, 1mM MgCl2, and 1.8mM CaCl2, pH7.4) with or without agonist or serial dilution of antagonist. After sealing the plates with HotSeal (Diversified Biotech, Boston, MA), the plates were incubated at 37°C for 15 min. Afterward, the plates were placed in a water bath at 85 to 90°C for 5min so as to lyse the cells and to release the intracellular cAMP. After centrifuging the plates at 500g for 2min, the amount of cAMP in 4μl of the supernatant was determined with the AlphaScreen cAMP detection kit (BioSignal, Montreal, QC, Canada) according to the manufacturer’s instructions, luminescence was measured with the α-Fusion (PerkinElmer Life Sciences) plate reader.
Cells were transiently co-transfected with either pCMX-Gal4 or pGal4-CRFR1/mutated CRFR1 with pFR-luc reporter. 24h post transfection, cells were washed once with 1x PBS, and lysed in 80μl/6-well plate 1x luciferase reporter lysis buffer (Promega, Madison, WI). 20μg protein lysates was mixed with 100 μl of luciferase assay reagent and relative light units measured using a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA).
This experiment was performed independently with the same parameters and normalized results were pooled. Oneway analysis of variance and post hoc Bonferroni multiple comparison test were performed using GraphPad Prism 5 software to analyze difference among the means of the normalized results. Differences with P-value less than 0.05 were considered statistically significant.
CD44 mediated microglia activation following chronic morphine treatment
It has been recognized that CD44 could act as an activation molecule to elicit inflammation (45-46). In the present study, by immunofluorescent assay, Fig.1A and B illustrated that CD44 expression was strongly enhanced following chronic morphine treatment and subsequent naloxone precipitation. Moreover, it was revealed that CD44 up-regulation occurred in cultured microglia but not in neuron or astrocyte (Fig.1C, D). By ELISA assay, it was further illustrated that, the production of CD44 related cytokines (IL-1β, iNOS, MCP-1, MIPα, MIPβ, and TNF-α) in prefrontal cortex, were considerably strengthened following chronic morphine treatment and subsequent naloxone replacement (Fig.1E). Notably, this up-regulation was observed in microglia but not in neuron or astrocyte (Fig.1F-H). In the same time, in neurons, receptors for above cytokines (IL-RI, CCR2, CCR3, CCR5, TLR-4) were evidently enhanced, the values rose around 2.5-2.7 folds over control (Fig.1I). Therefore, it was perceived that CD44 played a crucial role in the recruitment of microglia following chronic morphine exposure, which might initiate communication between neuron and microglia.
Activation of CRFR1 by c-Src signaling following longterm morphine treatment
In previous study, we characterized that Src kinases activation is a prerequisite for morphine-induced AC superactivation. Herein, in primary cell culture, it was illustrated that c-Src activity in neuron but not glial cells, was considerably enhanced after chronic morphine exposure and subsequent naloxone precipitation, [γ-32P] incorporation was increased around 3.1 folds over control, especially, the enhancement was strengthened when exposed to cytokines (Fig.2A). Coincidently, by Western Blot analysis, c-Src expression was initiated by 4h of morphine treatment and subsequent naloxone addition, the effect was concentrated in cytosol of neurons (Fig.2B, C).
In the same time, RSK1 expression and connection of RSK1 and CRFR1 were measured by Western Blot and immunoprecipitation respectively, as illustrated in Fig.1D and E, immune- positive signals for RSK1 and CRFR1 in RSK1 positive pool were considerably up-regulated following 4h of morphine treatment and subsequent naloxone precipitation.
CRFR1 phophorylation following chronic morphine treatment
By immunofluorescence, Fig.3A illustrated that pCRFR1 expression in prefrontal cortex was greatly up-regulated in morphine tolerant mice. Further by Western Blot analysis, CRFR1 phosphorylation was confirmed, the immune-positive signals were increased around 2.5 folds over control, and the enhancement could be strengthened by cytokines exposure (Fig.3B, C). N2A cells expressing CRFR1 or several mutated CRFR1-S372A, -S386A, -S396A, -S400A, -S405A, -S412A were established, by luciferase assay, it was revealed that only CRFR1-S396A activity was dependent on RSK-1 (Fig.3D). We also evaluated the role of CRFR1 on cAMP production in cortical neuron, as illustrated, intracellular cAMP level was increased in response to CRF exposure, the effect could be inhibited by CRF antibody, interestingly, the up-regulation of intracellular cAMP level could be strengthened by cytokines exposure (Fig.3E).
Figure 1. CD44 mediated microglia activation following chronic morphine treatment. A and B, mice were undergone chronic morphine treatment and naloxone precipitation, cross section of prefrontal cortex were mounted, and immune-stained with anti-CD44 Alexa 594 antibody, each group were analyzed by Leika Q500IW image analysis system, scale bar=50μm. C and D, neuron, astrocyte and microglia from prefrontal cortex were cultured, CD44 expression was measured by Western Blot analysis. E, mice were undergone chronic morphine treatment and naloxone precipitation, prefrontal cortex was separated, production of CD44 related cytokines including IL-1β, iNOS, MCP-1, MIP-α, MIP-β, TNF-α was detected by ELISA assay. F-H, neuron, astrocyte and microglia from prefrontal cortex were cultured, production of CD44 related cytokines including IL-1β, iNOS, MCP-1, MIP-α, MIP-β, TNF-α was detected by ELISA assay. I, neuron was cocultured with microglia in the presence of morphine for the indicated time, production of IL-1RI, CCR2, CCR3, CCR5, TNFR was measured by ELISA assay. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M of five independent experiments.
*p<0.05 vs saline or control.
Figure 2. Activation of CRFR1 by c-Src signaling following long-term morphine treatment. A, neuron, astrocyte and microglia from prefrontal cortex were cultured, in the presence or absence of cytokines, Src activity was detected by in vitro kinase assay. Cortical neurons were grown for 10 days, then exposed to chronic morphine treatment and subsequent naloxone replacement in the presence or absence of cytokines. B and C, cytosol and nucleus were separated and c-Src expression was measured by Western Blot analysis; D and E, RSK1 expression was detected by Western Blot analysis, connection of RSK1 and CRFR1 was detected by immnoprecipitation, in which anti-RSK1 was use as an immunoprecipitated antibody and anti-CRFR1 as an immunoblot antibody. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M of five independent experiments. *p<0.05 vs control.
Figure 3. CRFR1 phophorylation following chronic morphine treatment. Mice were undergone chronic morphine treatment and naloxone precipitation. A, cross section of prefrontal cortex were mounted, and immune-stained with anti-pCRFR1 and Alexa 488 antibody, each group were analyzed by Leika Q500IW image analysis system, scale bar=50μm; B and C, prefrontal cortex was separated and pCRFR1 expression was analyzed by Western Blot analysis. D, N2A cells expressing CRFR1 or several mutated CRFR1-S372A, -S386A, -S396A, -S400A, -S405A, -S412A were established, RSK siRNA was transiently transfected into cells followed by 4h of morphine treatment and subsequent naloxone precipitation, CRFR1 activity was measured by luciferase assay. E, cortical neuron was cultured in the presence of CRF or CRF antibody, amount of intracellular cAMP production in the presence of 10μM forskolin was measured using AlphaScreen cAMP detection kit. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M of five independent experiments. *p<0.05 vs control.
Disconnection of RGS4 and CRFR1 following chronic morphine treatment
RGS4 is a heterotrimeric G-protein inhibitor [42-43]. By immunoprecipitation in which anti-CRFR1 was used as immunoprecipitated antibody and anti-RGS4 as immunoblot antibody, it was illustrated that connection of CRFR1 and RGS4 was disrupted in prefrontal cortex in morphine tolerant mice, the inhibition could be deteriorated by cytokines exposure (Fig.4A). Coincidently, above alterations were concentrated in neuron (Fig.4B). Additionally, in N2A cells expressing MOR and CRFR1, intracellular cAMP levels was firstly attenuated by 5min of morphine treatment, and then increased to around 100% and 281.1±21.3 % control after 4 h of morphine exposure and subsequent naloxone precipitation, this cAMP production exhibited significant inhibition after over-expression of RGS4. Comparably, in N2A cells expressing MOR and CRFR1-S396A, only cAMP production mediated by chronic morphine treatment was interrupted by RGS4 over-expression (Fig.4C).
Figure 4. Disconnection of RGS4 and CRFR1 following chronic morphine treatment. A, mice were undergone chronic morphine treatment and naloxone precipitation, prefrontal cortex was separated, connection of CRFR1 and RGS4 was detected by immnoprecipitation, in which anti-CRFR1 was use as an immunoprecipitated antibody and anti-RGS4 as an immunoblot antibody; B, cortical neurons were grown for 10 days, then exposed to chronic morphine treatment and subsequent naloxone replacement in the presence or absence of cytokines, connection of CRFR1 and RGS4 was detected by immnoprecipitation, in which anti-CRFR1 was use as an immunoprecipitated antibody and anti-RGS4 as an immunoblot antibody. C, N2A cells expressing CRFR1 or CRFR1-S396A was transiently transfected with RGS4 plasmid followed by 4h of morphine treatment and subsequent naloxone precipitation, amount of intracellular cAMP production in the presence of 10μM forskolin was measured using AlphaScreen cAMP detection kit. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M. of three independent experiments. *p<0.05 vs control or saline.
Recruitment of RGS4 into MOR signaling following chronic morphine treatment
Above results promoted to study the functional consequence of RGS4 on long-term morphine responses. By immunoprecipitation
in which anti-MOR was used as immunoprecipitated antibody and anti-RGS4 as immunoblot antibody, it was illustrated that connection of MOR and RGS4 was increased in prefrontal cortex in morphine tolerant mice, the effect could be strengthened by cytokines exposure (Fig.5A). In cortical neuron, enhanced connection of MOR and RGS4 by chronic morphine was confirmed by BRET assay (Fig.5B). Meanwhile, [35S]GTPγS binding was considerably suppressed after long-term morphine treatment and naloxone precipitation, which was highly deteriorated when RGS4 over-expression or cytokines exposure ( Fig.5C). Additionally, in this process, there was no detectable alteration in CRF release from prefrontal cortex or CRF expression in neuron (Fig.5D-F).
Influence of cytokines on MOR signaling following chronic morphine treatment
By Western Blot analysis, it was found that AC5 expression in N2A cells expressing MOR/CRFR1 but not MOR/CRFR1- S396A, was dramatically increased by long-term morphine treatment and subsequent naloxone replacement, as expected, the effect could be enhanced by cytokines exposure (Fig.6A, B). Similar alteration was observed in cAMP production (Fig.6C). Moreover, by Western Blot analysis, it was demonstrated that AC5 expression in synaptoneurosome of prefrontal cortex was increased around 2.6 folds over control following chronic morphine treatment and subsequent naloxone precipitation, the preference expression could be strengthened by CRFR1 over-expression and knockdown by CRFR1 siRNA (Fig.6D, E).
Figure 5. Recruitment of RGS4 into MOR signaling following chronic morphine treatment. A, mice were undergone chronic morphine treatment and naloxone precipitation, prefrontal cortex was separated, connection of MOR and RGS4 was detected by immnoprecipitation, in which anti-MOR was use as an immunoprecipitated antibody and anti-RGS4 as an immunoblot antibody. B, cortical neuron was transiently co-transfected with MOR-Rluc and GFP2-RGS4 or GFP2 vector followed by chronic morphine treatment and subsequent naloxone precipitation, reading of the signals detected in the 370–450- and 500–530-nm windows, the BRET signal was determined by the ratio of the light emitted by the GFP2 or GFP2-RGS4 (500–530 nm) over the light emitted by the MOR-Rluc (370–450 nm). C, cortical neurons were grown for 10 days, then undergone chronic morphine treatment and naloxone precipitation in the presence or absence of cytokines, GTPγS binding was performed to assay the coupling of MOR and Gαi2. D, mice were undergone chronic morphine treatment and naloxone precipitation, CRF release from prefrontal cortex was detected by HPLC. E and F, cortical neurons were grown for 10 days, then undergone chronic morphine treatment and naloxone precipitation in the presence or absence of cytokines, CRF expression was detected by Western Blot analysis. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M of five independent experiments. *p<0.05 vs control.
Figure 6. Influence of cytokines on MOR signaling following chronic morphine treatment. N2A-MOR expressing CRFR1/CRFR1-S396A, was exposed to 4h of morphine treatment and subsequent naloxone replacement. A and B, AC5 expression was detected by Western Blot analysis; C, amount of intracellular cAMP production in the presence of 10μM forskolin was measured using AlphaScreen cAMP detection kit. D and E, mice were undergone chronic morphine treatment and naloxone precipitation in the presence or absence of cytokines, synaptoneurosome was separated from prefrontal cortex, AC5 expression was detected by Western Blot analysis. Data are normalized and calculated as percentage of control, each value represents mean±S.E.M of five independent experiments. *p<0.05 vs control or saline.
Figure 7. Behavioral effects of CRFR1 on morphine dependence and tolerance. Mice were undergone chronic morphine treatment and naloxone precipitation in the presence or CRFR1 or mutated CRFR1, control group received vehicle solution or blunt vector (n=5): A, mice were placed in the indicated monitor, withdrawal scores including jumping, tremor, wet-dog shake, rear and grooming were measured based on individual signs; B, mice were subjected to hot-plate, the cutoff time was set at 10s, antinociceptive response is expressed as the mean with S.E.M. of percentage of the maximum possible effect (%MPE). C, mice were placed in the NE quadrant of the water maze without the platform for 60s, the amount of time the mice spent in each quadrant searching for the platform was recorded. Probe tests were performed every 4 days afterward until the mice spent an equal amount of time in each quadrant. Data are normalized and calculated, each value represents mean±S.E.M of five independent experiments. *p<0.05 vs saline.
Behavioral effects of CRFR1 on morphine dependence and tolerance
By counting somatic withdrawal signs including jumps, tremors, wet-dog shakes, rears, and grooming behavior, Fig.7A illustrated that, all monitored withdrawal signs were greater in long-term morphine treatment than control animals (jumps: t=10.5, P<0.001; tremors: t=5.25, P<0.001; wet-dog shakes: t=2.75, P<0.05; rears: t=16.2, P<0.001; grooming: t=18.9, P<0.001), these increased number of withdrawal signs could be alleviated by over-expression of CRFR1-S396A, instead, mice with CRFR1 over-expression showed higher number of withdrawal signs (jumping, rear and grooming) than morphine treatment regimen.
Antinociceptive property of morphine was examined by hotplate test, a paradigm that primarily assessed supraspinal pain responsiveness . It was demonstrated that the onset of morphine tolerance was readily apparent in mice after 2 days of morphine treatment, the degree of tolerance was sustained even in the presence of CRFR1 or CRFR1-S396A (Fig.7B). Mice were placed in the NE quadrant of the water maze without the platform for 60s, the amount of time the mice spent in each quadrant searching for the platform was recorded. It was observed that mice spent less time in SW quadrant following chronic morphine treatment, this performance could be deteriorated and alleviated with over- or down-regulation of CRFR1 (Fig.7C).
Currently, we demonstrated that microglia activation appeared to be obligatory for CD44 expression and production of IL-1β, iNOS, MCP-1, MIP-α, MIP-β, and TNF-α, which was primarily initiated by by long-term morphine treatment and subsequent naloxone precipitation. Well characterized, CD44 is a transmembrane glycoprotein that plays a critical role in a variety of cellular behaviors (26, 46). Mounting evidence has established that neuro-inflammation is associated with increased expression of CD44 in cell surface, which can mediate microglia adhesion, migration and invasion  . Accordingly, our data added diversity to the range of potential functions of microglia activation, and supported the idea that neuroinflammation polymorphisms might be the risk for morphine tolerance and dependence.
Previous studies showed that Src kinases could be recruited to opioid receptor following chronic morphine treatment, which resulted in an additional significant increase of cAMP signaling [17-18]. Herein, we observed similar Src kinase activation following long-term morphine treatment, notably, this Src kinase activation was preferentially concentrated in neuron and distinctively lead to CRFR1 phosphorylation, especially, above alteration was remarkably sensitized in the presence of microglia activation. Mounting evidence indicated that the negative states of withdrawal mostly involve recruitment of brain stress neurocircuitry, including induction of hypothalamo-pituitary-adrenocortical (HPA) axis, noradrenergic activity, and corticotropin-releasing factor (CRF) activity [37-41]. Concordant with this view, CRFR1 phosphorylation at serine 396 might provide an important biochemical substratum for morphine mediated cAMP production.
At present, evidence implicates that the duration and amplitude of G-protein-coupled receptor (GPCR) signaling is controlled by regulator of G-protein signaling (RGS) proteins. RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartment is demonstrated to have regulatory roles in modulation of MOR activity and several stress responses [42-43]. In the present study, we found that chronic morphine evoked a signaling shift from MOR to CRFR1 via RGS4, and thereby resulted in MOR-G protein inhibition and cAMP production, also, above alteration could be enhanced in the presence of microglia activation. Moreover, AC5 expression was recognized to be under the control of CRFR1 phophsorylation and could be strengthened in the presence of microglia activation. Adding to this, we demonstrated further that AC5 up-regulation could be profoundly concentrated in synaptosome. Then, the present finding raised the possibility that the spatial and temporal regulation of AC5 activation was mostly related with altered neuronal and synaptic architecture, dendritic morphology and axonal branching, which was thought to be circuit adaptations for long-lasting effects of morphine [48-53]. As such, it was very likely that microglia activation launched neuronal responses that were prerequisite for creating a state of morphine dependence.
Of most interest, morphine produced expression of somatic withdrawal signs including jumping, tremor, wet-dog shake, rear and grooming, which could be relieved by CRFR1 modulation. Similar effect could be observed in place conditioning but not antinociceptive tolerance. Therefore, the observation was apparently extended to the realization that functional desensitization of MOR appeared to predominantly under the control of neuron-microglia communication, signaling shift from MOR to CRFR1 via RGS4 may be one element of the neurobiological mechanisms for morphine withdrawal .
Our current studies demonstrated that following long-term morphine treatment and subsequent naloxone precipitation, microglia was activated, which appeared to be obligatory for CD44 expression and production of IL-1β, iNOS, MCP- 1, MIP-α, MIP-β, and TNF-α. Significantly, we demonstrated that following prolonged morphine exposure, c-Src was up-regulated in neuron and subjected to microglia activation. Moreover, it was identified that CRFR1 could be phosphorylated at serine 396 by c-Src-RSK1 signaling, the phosphorylation was profoundly evoked by microglia activation, and preferentially promoted recruitment of RGS4 into MOR signaling complex, converting MOR activation into MOR-G protein inhibition and cAMP production. Adding to this, AC5
up-regulation was demonstrated to be strengthened and predominantly under the control of CRFR1 phosphorylation. Significantly, RGS4 delivered signaling shift from MOR to CRFR1, which was involved in morphine produced expression of somatic withdrawal signs and place conditioning but not antinociceptive tolerance. Thus, the data indicated that desensitization of MOR appeared to predominantly under the control of neuron-microglia communication, the sensitized brain CRFR1 receptor circuitry served to maintain and potentiate neuro-adaptive morphine activities.
This research was supported by grants from National Nature Sciences of China (81471370), and National Key Basic Research Program of China (2013CB531900).
The authors declare that they have no competing interests.
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