Objective: To explore the effects of the mu-opioid receptor (MOR) in the paraventricular nucleus (PVN) on the ejaculatory behaviors of male rats and its potential mechanisms. METHODS: Male SD rats with normal ejaculation ability were mated with female ones in hormone-induced estrus. After bilateral PVN microinjection of D-Ala-2-Me-Phe-4-Gly-ol enkephalin (DAGO) or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) with an inserted catheter, the male animals were observed for mount latency (ML), mount frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), ejaculation frequency (EF), post-ejaculation interval (PEI), and intromission ratio (IR). The lumbar sympathetic nerve activity (LSNA) of the rats was recorded using the PowerLab data acquisition hardware device, and the levels of norepinephrine (NE) in the peripheral plasma were measured by ELISA following microinjection of saline or different doses of DAGO or CTAP. RESULTS: Neither CTAP nor DGAO significantly affected the ML of the male rats (P > 0.05). DGAO remarkably increased IF (P < 0.01) and MF (P < 0.01), prolonged IL (P < 0.01), EL (P < 0.01) and PEI (P < 0.01), and reduced EF (P <0.01) and IR (P < 0.05). On the contrary, CTAP markedly decreased IF (P < 0.01) and MF (P < 0.01), shortened IL (P < 0.01), EL (P < 0.01) and PFI (P < 0.01), and elevated EF (P < 0.01) and IR (P < 0.01). Additionally, DAGO decreased LSNA in a dose-dependent manner and reduced the NE level in the peripheral plasma. CTAP, however, not only offset the effects of DAGO on LSNA, but also significantly increased LSNA. CONCLUSIONS MOR in PVN inhibits ejaculatory behaviors in male rats by weakening LSNA, which has provided some theoretical evidence for the use of highly selective opioids in the treatment of premature ejaculation.
Animals ; Ejaculation ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology* ; Female ; Male ; Paraventricular Hypothalamic Nucleus/physiology* ; Peptide Fragments/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, mu/physiology* ; Somatostatin/pharmacology* ; Sympathetic Nervous System/physiology*

µ-opioid receptor (MOR) is a class of opioid receptors with a high affinity for enkephalins and beta-endorphin. In hippocampus, activation of MOR is known to enhance the neuronal excitability of pyramidal neurons, which has been mainly attributed to a disinhibition of pyramidal neurons via activating Gαi subunit to suppress the presynaptic release of GABA in hippocampal interneurons. In contrast, the potential role of MOR in hippocampal astrocytes, the most abundant cell type in the brain, has remained unexplored. Here, we determine the cellular and subcellular distribution of MOR in different cell types of the hippocampus by utilizing MOR-mCherry mice and two different antibodies against MOR. Consistent with previous findings, we demonstrate that MOR expression in the CA1 pyramidal layer is co-localized with axon terminals from GABAergic inhibitory neurons but not with soma of pyramidal neurons. More importantly, we demonstrate that MOR is highly expressed in CA1 hippocampal astrocytes. The ultrastructural analysis further demonstrates that the astrocytic MOR is localized in soma and processes, but not in microdomains near synapses. Lastly, we demonstrate that astrocytes in ventral tegmental area and nucleus accumbens also express MOR. Our results provide the unprecedented evidence for the presence of MOR in astrocytes, implicating potential roles of astrocytic MOR in addictive behaviors.
Animals ; Antibodies ; Astrocytes* ; Behavior, Addictive ; beta-Endorphin ; Brain ; Carisoprodol ; Enkephalins ; gamma-Aminobutyric Acid ; Hippocampus ; Interneurons ; Mice ; Microscopy, Electron ; Neurons ; Nucleus Accumbens ; Presynaptic Terminals ; Pyramidal Cells ; Receptors, Opioid ; Synapses ; Ventral Tegmental Area

The superficial dorsal horn of the spinal cord plays an important role in pain transmission and opioid activity. Several studies have demonstrated that opioids modulate pain transmission, and the activation of µ-opioid receptors (MORs) by opioids contributes to analgesic effects in the spinal cord. However, the effect of the activation of MORs on GABAergic interneurons and the contribution to the analgesic effect are much less clear. In this study, using transgenic mice, which allow the identification of GABAergic interneurons, we investigated how the activation of MORs affects the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive afferent and GABAergic interneurons. We found that a selective µ-opioid agonist, [D-Ala², NMe-Phe⁴, Gly-ol]-enkephanlin (DAMGO), induced an outward current mediated by K⁺ channels in GABAergic interneurons. In addition, DAMGO reduced the amplitude of evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons which receive monosynaptic inputs from primary nociceptive C fibers. Taken together, we found that DAMGO reduced the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive C fibers and GABAergic interneurons. These results suggest one possibility that suppression of GABAergic interneurons by DMAGO may reduce the inhibition on secondary GABAergic interneurons, which increase the inhibition of the secondary GABAergic interneurons to excitatory neurons in the spinal dorsal horn. In this circumstance, the sum of excitation of the entire spinal network will control the pain transmission.
Analgesics, Opioid ; Animals ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Excitatory Postsynaptic Potentials ; GABAergic Neurons* ; Interneurons ; Mice ; Mice, Transgenic ; Nerve Fibers, Unmyelinated ; Neurons ; Spinal Cord ; Spinal Cord Dorsal Horn* ; Substantia Gelatinosa ; Synaptic Transmission

OBJECTIVETo observe analgesic effect of electroacupuncture ( EA) on rats with chronic inflammatory pain and its regulatory mechanism on ispilateral dorsal root ganglion (DRG) and spinal dorsal horn (SDH) Mas-related G protein-coupled C receptor (MrgprC).

METHODSTotally 40 healthy male SD rats were divided into 4 groups according to random number table, i.e., the normal (N) group, the model (M) group, the acupuncture (Acu) group, the EA group, 10 rats in each group. The model of chronic inflammatory pain was established by subcutaneous injecting 0. 1 mL complete Freund's adjuvant (CFA) into right hind paw. Paw withdrawal thresholds (PWTs) were measured before modeling, at day 1, 3, 5, 7, and after CFA injection, respectively. Expression levels of MrgprC in ispilateral DRG and SDH were detected by Western blot. The content of bovine adrenal medulla 22 (BAM22) in SDH was detected by immunohistochemical assay.

RESULTSCompared with N group at each time point, PWTs significantly decreased in M group (P <0. 01). Compared with M group, PWTs significantly increased at day 5 of EA and after EA in EA group (P < 0.05, P < 0.01). Compared with Acu group at each time point, post-EA PWTs significantly increased in the EA group (P < 0.05). Compared with N group, expression of MrgprC in ispilateral DRG and ratio of BAM22 positive cells in ispilateral SDH increased in M group (P < 0.01). Compared with M group, expression of MrgprC in ispilateral DRG and ratio of BAM22 positive cells in ispilateral SDH increased in the EA group (P < 0.05).

CONCLUSIONEA had favorable analgesic effect on chronic inflammatory pain induced by CFA, and its mechanism might be possibly associated with up-regulating MrgprC expression in ispilateral DRG and BAM22 content in ispilateral SDH.

Analgesia ; Animals ; Electroacupuncture ; Enkephalins ; metabolism ; Freund's Adjuvant ; Ganglia, Spinal ; drug effects ; Inflammation ; chemically induced ; drug therapy ; Male ; Pain Management ; methods ; Peptide Fragments ; metabolism ; Posterior Horn Cells ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the analgesic mechanism of small knife needle for treating transverse process syndrome of the third vertebra (TPSTV) by observing peripheral and central changesof β-endorphin (β-EP) and enkephalin (ENK) contents.

METHODSTotally 30 Japanese white big-ear rabbits of clean grade were divided into 5 groups according to random digit table, i.e., the normal control group, the model group, the small knife needle group, the electroacupunture (EA) group, and the small knife needle plus EA group, 6 in each group. The TPSTV model was established by inserting a piece of gelatin sponge into the left transverse process of 3rd lumbar vertebrae. Rabbits in the small knife needlegroup were intervened by small knife needle. Those in the EA group were intervened by EA at bilateralWeizhong (BL40). Those in the small knife needle plus EA group were intervened by small knife needleand EA at bilateral Weizhong (BL40). Contents of β-EP and ENK in plasma, muscle, spinal cord, and hypothalamus were determined after sample collection at day 28 after modeling.

RESULTSCompared with the normal control group, contents of β-EP and ENK in plasma and muscle increased significantly, and contents of β-EP and ENK in spinal cord and hypothalamus decreased significantly in the model group (P < 0.05, P < 0.01). Contents of β-EP and ENK approximated normal levels in the three treatment groups after respective treatment. Compared with the model group, the content of β-EP in muscle decreased, and contents of β-EP and ENK in hypothalamus increased in the three treatment groups after respective treatment (P < 0.05). There were no significant difference among the three treatment groups (P > 0.05).

CONCLUSIONSSmall knife needle treatment and EA had benign regulation on peripheral and central β-EP and ENK in TPSTV rabbits. Small knife needle treatment showed better effect than that of EA.

Acupuncture Points ; Animals ; Electroacupuncture ; Enkephalins ; metabolism ; Hypothalamus ; metabolism ; Lumbar Vertebrae ; pathology ; Muscle, Skeletal ; metabolism ; Needles ; Rabbits ; Random Allocation ; Spinal Cord ; metabolism ; Spinal Diseases ; therapy ; beta-Endorphin ; metabolism

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X−/−) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X−/−) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.
Analgesia* ; Animals ; Brain ; Codon, Nonsense ; Dopamine ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Ethylnitrosourea ; Fertilization in Vitro ; Gene Library ; Mass Screening ; Mice ; Morphine* ; Phosphotransferases ; Protein Tyrosine Phosphatases ; Receptors, Opioid ; Reward* ; Street Drugs ; Substance Withdrawal Syndrome*

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K⁺ current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K⁺ channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K⁺ equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K⁺ channel) related acid-sensitive K⁺ channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K⁺ current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K⁺ channel (TASK1 and 3) in addition to inwardly rectifying K⁺ channel.
Analgesics, Opioid ; Animals ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-* ; Hydrogen-Ion Concentration ; Membrane Potentials ; Neurons* ; Rats* ; RNA, Messenger ; Spinal Cord* ; Substantia Gelatinosa*

Learning/memory impairment is one of the most serious problems induced by stress, and the underlying mechanisms remain unclear. Opiates and opioid receptors are implicated in multiple physiological functions including learning and memory. However, there is no clear evidence whether the endogenous opioid system is involved in the formation of the stress-induced spatial reference memory impairment. The aim of the present study was to evaluate the role of μ opioid receptor in the stress-induced spatial reference memory impairment by means of Morris water maze (MWM) test in a mouse elevated platform stress model. The mice were trained in the MWM for four trials a session for 4 consecutive days after receiving the elevated platform stress, and intracerebroventricular injection of μ opioid receptor agonist DAMGO, antagonist CTAP or saline. Retention of the spatial training was assessed 24 h after the last training session with a 60-s free-swim probe trial using a new starting position. The results showed that intracerebroventricular injection of μ opioid receptor agonist DAMGO but not antagonist CTAP before MWM training impaired the memory retrieval of mice. Elevated platform stress before MWM training also impaired memory retrieval, which could be reversed by pre-injection of CTAP, and aggravated by DAMGO. These results suggest that endogenous opioid system may play a crucial role in the formation of the stress-induced memory impairment.
Animals ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; pharmacology ; Maze Learning ; Memory Disorders ; Mice ; Receptors, Opioid, mu ; physiology ; Spatial Memory ; Stress, Physiological

Previous studies have reported the association of prodynorphin (PDYN) promoter polymorphism with temporal lobe epilepsy (TLE) susceptibility, but the results remain inconclusive. To further precisely evaluate this association, we performed a meta-analysis. Published studies of TLE and PDYN polymorphism up to February 2015 were identified. Subgroup analysis by TLE subtype was performed. Moreover, sensitivity, heterogeneity, and publication bias were also analyzed. Seven case-control studies were finally included in this meta-analysis with 875 TLE cases and 1426 controls. We did not find synthetic evidence of association between PDYN promoter polymorphism and TLE susceptibility (OR=1.184, 95% CI: 0.873-1.606, P=0.277). Similar results were also obtained in non-familial-risk TLE subgroup. However, in the familial-risk TLE subgroup analysis, a significant association was observed (OR=1.739, 95% CI: 1.154-2.619, P=0.008). In summary, this meta-analysis suggests that PDYN gene promoter polymorphism might contribute to familial-risk TLE.
Case-Control Studies ; Enkephalins ; genetics ; Epilepsy, Temporal Lobe ; diagnosis ; genetics ; pathology ; Family ; Gene Expression ; Genetic Association Studies ; Genetic Predisposition to Disease ; Humans ; Inheritance Patterns ; Odds Ratio ; Polymorphism, Genetic ; Prognosis ; Promoter Regions, Genetic ; Protein Precursors ; genetics

OBJECTIVETo observe the intervention of electroacupuncture (EA) with different current frequencies and treatment frequencies on pain thresholt in rats with bone-cancer pain, so as to optimize treatment parameters of EA against bone cancer pain; and by measuring gene expression of opioid receptor and precursor in different tissues to preliminarily explore the possible mechanism of EA against bone cancer pain.

METHODSNinety healthy female SD rats were randomly divided into a control group, a model group, EA groups (6 subgroups according to different frequencies) and a sham EA group, ten rats in each one. Rats in the control group were injected with 10 µL of amicrobic phosphate buffer solution (PBS) into tibial cavity; rats in the remaining groups were injected with Walker 256 cancer cells to establish model of bone-cancer pain. No treatment was given to rats in the control group and model group; rats in the EA groups were treated with EA at bilateral "Housanli" (ST 36) and "Genduan" with 3 different current frequencies (2 Hz, 100 Hz and 2 Hz/100 Hz), once a day and once every other day, 30 min per treatment (1mA for 15 min, 2 mA for 15 min); rats in the sham EA group were treated with identical acupoints as the EA group, but the acupoints were needled subcutaneously and EA was connected with power off. All the treatment was given for 14 days. Dynamic plantar aesthesiometer was applied to measure the paw withdrawal thresholds (PWTs) of the affected side before the model establishment, 6d, 8d, 10d, 12d, 14d, 16d, 18d, and 20d after model establishment. The mRNA expressions of µ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), proopiomelanocortin (POMC) and prodynorphin (PDYN) in dorsal root ganglion (DRG) and lumbar spinal cord dorsal horn (SCDH) of L4-L6 of the affected side were detected by PCR method.

RESULTSThere were no differences in PWTs among all groups before model establishment (P>0. 05). Each time point after model establishment, PWTs in model group were obviously lower than those in the control group (all P<0. 01). Compared with the model group, PWTs in each EA subgroup were all increased (all P<0.05), but the differences at different time points were not significant among EA subgroups (P>0.05). The mRNA expressions of MOR, KOR, POMC, and PDYN in L4-L6 DRG in the 2 Hz/100 Hz II group were significantly higher than those in model group (P<0. 05, P<0. 01), while the mRNA expressions of MOR, KOR, DOR, POMC and PDYN in SCDH were not different compared with the model group (P>0. 05).

CONCLUSIONEA treatment has obvious analgesic effect on bone-cancer pain, however, its effect is not related with current frequency and treating frequency. EA against bone-cancer pain may be related with increasing the mRNA expression of some peripheral opioid receptors and precursor.

Acupuncture Analgesia ; instrumentation ; methods ; Acupuncture Points ; Animals ; Bone Neoplasms ; complications ; Electroacupuncture ; instrumentation ; methods ; Enkephalins ; metabolism ; Female ; Ganglia, Spinal ; metabolism ; Humans ; Pain ; etiology ; genetics ; metabolism ; Pain Management ; instrumentation ; methods ; Protein Precursors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; genetics ; metabolism