enkephalin--ala(2)-mephe(4)-gly(5)- and Substance-Withdrawal-Syndrome

Opioid drugs are a first-line treatment for severe acute pain and other chronic pain conditions, but long-term opioid drug use produces opioid-induced hyperalgesia (OIH). Co-administration of cannabinoids with opioid receptor agonists produce anti-nociceptive synergy, but cannabinoid receptor agonists may also produce undesirable side effects. Therefore, positive allosteric modulators (PAM) of cannabinoid type-1 receptors (CB1R) may provide an option reducing pain and/or enhancing the anti-hyperalgesic effects of opioids without the side effects, tolerance, and dependence observed with the use of ligands that target the orthosteric binding sites. This study tested GAT211, a PAM of cannabinoid type-1 receptors (CB1R), for its ability to enhance the anti-hyperalgesic effects of the mu-opioid receptor (MOR) agonist DAMGO in rats treated chronically with morphine (or saline) and tested during withdrawal. We tested the effects of intra-periaqueductal gray (PAG) injections of (1) DAMGO, (2) GAT211, or (3) DAMGO + GAT211 on thermal nociception in chronic morphine-treated rats that were hyperalgesic and also in saline-treated control rats. We used slice electrophysiology to test the effects of DAMGO/GAT211 bath application on synaptic transmission in the vlPAG. Intra-PAG DAMGO infusions dose-dependently reversed chronic morphine-induced hyperalgesia, but intra-PAG GAT211 did not alter nociception at the doses we tested. When co-administered into the PAG, GAT211 antagonized the anti-nociceptive effects of DAMGO in morphine-withdrawn rats. DAMGO suppressed synaptic inhibition in the vlPAG of brain slices taken from saline- and morphine-treated rats, and GAT211 attenuated DAMGO-induced suppression of synaptic inhibition in vlPAG neurons via actions at CB1R. These findings show that positive allosteric modulation of CB1R antagonizes the behavioral and cellular effects of a MOR agonist in the PAG of rats.

Topics: Allosteric Regulation; Analgesics, Opioid; Animals; Cannabinoid Receptor Agonists; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hyperalgesia; Male; Microinjections; Morphine; Nociception; Periaqueductal Gray; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Substance Withdrawal Syndrome

Unlike the behavioral effects planarians display when exposed to cocaine, amphetamines, cathinones, ethanol and sucrose, effects of opioid receptor agonists, especially mu opioid receptor agonists, are poorly defined in these flatworms. Here, we tested the hypothesis that planarians exposed to a selective mu opioid receptor agonist, DAMGO (0.1, 1, 10 µM), would display a triad of opioid-like effects (place conditioning, abstinence-induced withdrawal, and motility changes). DAMGO was selected versus morphine because of its greater mu opioid receptor selectivity. In place conditioning and abstinence experiments, the planarian light/dark test (PLDT) was utilized (i.e., planarians are placed into a petri dish containing water that is split into light and dark compartments and time spent in the compartments is determined). Planarians conditioned with DAMGO (1 µM) spent more time on the drug-paired side compared to water controls. In abstinence experiments, planarians exposed to DAMGO for 30 min were removed and then placed into water, where light avoidance (e.g. defensive responding) and depressant-like effects (i.e., decreased motility) were quantified. Compared to water controls, DAMGO-withdrawn planarians spent less time in the light (10 µM) and displayed decreased motility (1, 10 µM). Acute DAMGO exposure (1 µM) produced hypermotility that was antagonized by naltrexone (1, 10, 100 µM). In contrast, acute exposure to the kappa opioid receptor agonist U50,488H (0.1, 1, 10 µM) resulted in decreased motility. Our results show that a mu opioid agonist produces mammalian-like behavioral responses in planarians that may be related to addiction and suggest opioid-like behavioral effects are conserved in invertebrates.

Topics: Analgesics, Opioid; Animals; Conditioning, Psychological; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Locomotion; Naltrexone; Narcotic Antagonists; Planarians; Receptors, Opioid, mu; Substance Withdrawal Syndrome

Protracted opiate withdrawal is accompanied by altered responsiveness of midbrain dopaminergic (DA) neurons, including a loss of DA cell response to morphine, and by behavioral alterations, including affective disorders. GABAergic neurons in the tail of the ventral tegmental area (tVTA), also called the rostromedial tegmental nucleus, are important for behavioral responses to opiates. We investigated the tVTA-VTA circuit in rats after chronic morphine exposure to determine whether tVTA neurons participate in the loss of opiate-induced disinhibition of VTA DA neurons observed during protracted withdrawal. In vivo recording revealed that VTA DA neurons, but not tVTA GABAergic neurons, are tolerant to morphine after 2 weeks of withdrawal. Optogenetic stimulation of tVTA neurons inhibited VTA DA neurons similarly in opiate-naive and long-term withdrawn rats. However, tVTA inactivation increased VTA DA activity in opiate-naive rats, but not in withdrawn rats, resembling the opiate tolerance effect in DA cells. Thus, although inhibitory control of DA neurons by tVTA is maintained during protracted withdrawal, the capacity for disinhibitory control is impaired. In addition, morphine withdrawal reduced both tVTA neural activity and tonic glutamatergic input to VTA DA neurons. We propose that these changes in glutamate and GABA inputs underlie the apparent tolerance of VTA DA neurons to opiates after chronic exposure. These alterations in the tVTA-VTA DA circuit could be an important factor in opiate tolerance and addiction. Moreover, the capacity of the tVTA to inhibit, but not disinhibit, DA cells after chronic opiate exposure may contribute to long-term negative affective states during withdrawal.. Dopaminergic (DA) cells of the ventral tegmental area (VTA) are the origin of a brain reward system and are critically involved in drug abuse. Morphine has long been known to affect VTA DA cells via GABAergic interneurons. Recently, GABAergic neurons caudal to the VTA were discovered and named the tail of VTA (tVTA). Here, we show that tVTA GABA neurons lose their capacity to disinhibit, but not to inhibit, VTA DA cells after chronic opiate exposure. The failure of disinhibition was associated with a loss of glutamatergic input to DA neurons after chronic morphine. These findings reveal mechanisms by which the tVTA may play a key role in long-term negative affective states during opiate withdrawal.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Afferent Pathways; Animals; Channelrhodopsins; Disease Models, Animal; Dopaminergic Neurons; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; GABAergic Neurons; Glutamate Decarboxylase; Male; Morphine; Narcotics; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Time Factors; Valine; Ventral Tegmental Area

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Calcitonin Gene-Related Peptide; Cell Line, Tumor; CREB-Binding Protein; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Hydrogen Sulfide; Hyperalgesia; Male; MAP Kinase Signaling System; Naloxone; Neuroblastoma; Pain Threshold; Rats; Rats, Sprague-Dawley; Spine; Substance Withdrawal Syndrome

Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia.

Topics: Analgesics, Opioid; Animals; Calcium; Capsaicin; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Diterpenes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Fentanyl; Ganglia, Spinal; Humans; Hyperalgesia; Male; Membrane Potentials; Morphine; Mutagenesis, Site-Directed; Protein Binding; Rats; Receptors, Opioid, mu; Sensory Receptor Cells; Substance Withdrawal Syndrome; Tritium; TRPV Cation Channels

μ-Opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive activity is highly relevant in the mouse VTA, as it regulates GABAergic input to dopamine neurons. Specifically, suppression of MOR constitutive activity with the inverse agonist KC-2-009 enhanced GABAergic neurotransmission onto VTA dopamine neurons. This inverse agonistic effect was fully blocked by the specific MOR neutral antagonist CTOP, which had no effect on GABAergic transmission itself. We next show that withdrawal from chronic morphine further increases the magnitude of inverse agonistic effects at the MOR, suggesting enhanced MOR constitutive activity. We demonstrate that this increase can be an adaptive response to the detrimental elevation in cAMP levels known to occur during morphine withdrawal. These findings offer important insights in the physiological occurrence and function of MOR constitutive activity, and have important implications for therapeutic strategies aimed at normalizing MOR signaling during addiction and opioid overdose.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Analysis of Variance; Animals; Colforsin; Cyclic AMP; Dopaminergic Neurons; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activators; Female; gamma-Aminobutyric Acid; Male; Mice; Mice, Inbred C57BL; Morphine; Pregnancy; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Ventral Tegmental Area

Clinical pain conditions may remain responsive to opiate analgesics for extended periods, but such persistent acute pain can undergo a transition to an opiate-resistant chronic pain state that becomes a much more serious clinical problem. To test the hypothesis that cellular mechanisms of chronic pain in the primary afferent also contribute to the development of opiate resistance, we used a recently developed model of the transition of from acute to chronic pain, hyperalgesic priming. Repeated intradermal administration of the potent and highly selective mu-opioid agonist, [d-Ala(2),N-MePhe(4),gly-ol]-enkephalin (DAMGO), to produce tolerance for its inhibition of prostaglandin E(2) hyperalgesia, simultaneously produced hyperalgesic priming. Conversely, injection of an inflammogen, carrageenan, used to produce priming produced DAMGO tolerance. Both effects were prevented by inhibition of protein kinase Cepsilon (PKCepsilon). Carrageenan also induced opioid dependence, manifest as mu-opioid receptor antagonist (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2))-induced hyperalgesia that, like priming, was PKCepsilon and G(i) dependent. These findings suggest that the transition from acute to chronic pain, and development of mu-opioid receptor tolerance and dependence may be linked by common cellular mechanisms in the primary afferent.

Topics: Acute Disease; Analgesics, Opioid; Animals; Carrageenan; Chronic Disease; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Hyperalgesia; Inflammation; Male; Nociceptors; Opioid-Related Disorders; Pain; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome

The functional activity of trimeric guanine-nucleotide-binding proteins (G-proteins) represents an essential step in linking and regulation of the opioid receptor (mu-, delta- and kappa-OR)-initiated signaling pathways. Theoretical basis and/or molecular mechanism(s) of opioid tolerance and addiction proceeding in the central nervous system were not studied in the forebrain cortex of mammals with respect to quantitative analysis of opioid-stimulated trimeric G-protein activity.. G-protein activity was measured in PercollR-purified plasma membranes (PM) isolated from the frontal brain cortex of control and morphine-treated rats by both high-affinity [32P]GTPase and [35S]GTPgammaS binding assays. Exposition to morphine was performed by intra-muscular application of this drug. Control animals were injected with sterile PBS.. Both mu-OR (DAMGO)- and delta-OR (DADLE)-responses were clearly desensitized in PM isolated from morphine-treated rats; kappa-OR (U-69593)- and baclofen (GABAB-R)-stimulated [35S]GTPgammaS binding was unchanged, indicating the specificity of the morphine effect. Under such conditions, the amount of G-protein alpha subunits was unchanged. The order of efficacy DADLE>DAMGO>U-69593 was the same in control and morphine-treated PM. Behavioral tests indicated that morphine-treated animals were fully drug-dependent and developed tolerance to subsequent drug addition.. Prolonged exposure of rats to high doses of morphine results in decrease of the over-all output of OR-stimulated G-protein activity in the forebrain cortex but does not decrease the amount of these regulatory proteins. These data support the view that the mechanism of the long-term adaptation to high doses of morphine is primarily based on desensitization of OR-response preferentially oriented to mu-OR and delta-OR.

Topics: Adaptation, Physiological; Animals; Baclofen; Behavior, Animal; Cell Membrane; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Male; Morphine; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Time Factors

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol-a GABA(A) receptors agonist-and DAMGO-a mu-opioid receptors agonist-on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence

Topics: Amygdala; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABA Agonists; GABA-A Receptor Agonists; gamma-Aminobutyric Acid; Microinjections; Morphine; Motor Activity; Muscimol; Narcotics; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, mu; Reflex, Startle; Substance Withdrawal Syndrome

The effect exerted by two gamma-endorphin derivatives (DTgammaE and DEgammaE) was investigated on morphine-induced inhibition on the electrically induced contractions of guinea pig ileum in vitro. Morphine (1x10(-8)-5x10 (-8)-1x10 (-7) M) dose dependently and significantly reduced the E.C. of guinea pig ileum, IC50=6.5x10(-8) M (Confidence limits: 3.7x10 (-8)-9.1x10 (-8)). DTgammaE and DEgammaE per se (1x10 (-6)-5x10 (-6)-1x10 (-5) M) did not modify significantly the E.C. of guinea pig ileum. Furthermore, DTgammaE or DEgammaE injection 10-30-60 min before morphine, did not affect the inhibitory effect of morphine on the E.C. of guinea pig ileum. By contrast, ilea from guinea-pigs treated for 4 days with DTgammaE or DEgammaE (1 mg/Kg/i.p.) were less sensitive to the inhibitory effect of morphine, IC50=8.3x10 (-7) M (Confidence limits: 1.4x10(-6)-3.5x10(-7)) for DTgammaE and IC50=7.7x10(-7) M (Confidence limits: 2.7x10(-6)-8.7x10(-7 )) for DEgammaE . The effect exerted by two beta-endorphin fragments (DTgammaE and DEgammaE) was investigated on the acute opiate withdrawal induced by micro, kappa and delta receptor agonists in vitro. After a exposure in vitro for 4 min to morphine (less selective micro agonist), DAGO (highly selective micro agonist), U50-488H (highly selective kappa agonist) and beta-endorphin (selective micro- delta agonist), a strong contracture of isolated guinea pig ileum was observed after the addition of naloxone. This effect was also observed when isolated rabbit jejunum was pretreated with deltorphin (highly selective delta agonist). DTgammaE or DEgammaE injection before or after treatment with morphine, DAGO, U50-488H, beta-endorphin or deltorphin was able of both preventing and reversing the naloxone-induced contracture after exposure to the opioid agonists in a concentration-dependent fashion. Our results indicate that both DTgammaE or DEgammaE are able to reduce significantly opiate withdrawal in vitro, suggesting an important functional interaction beween beta-endorphin fragments and opioid withdrawal at both micro, kappa and delta receptor level. Our results indicate that chronic treatment of guinea pigs with DTgammaE or DEgammaE induces a significant reduction of the inhibitory effect of morphine on the E.C. of guinea-pig ileum thus confirming an important functional interaction between gamma-endorphin derivatives and opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acetylcholine; Animals; beta-Endorphin; Electric Stimulation; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; In Vitro Techniques; Jejunum; Male; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Oligopeptides; Peptide Fragments; Rabbits; Substance Withdrawal Syndrome

Earlier studies suggest that opioid receptors in the ventral tegmental area, but not the nucleus accumbens (NAc), play a role in relapse to drug-seeking behavior. However, environmental stimuli that elicit relapse also release the endogenous opioid beta-endorphin in the NAc. Using a within-session extinction/reinstatement paradigm in rats that self-administer cocaine, we found that NAc infusions of the mu-opioid receptor (MOR) agonist DAMGO moderately reinstated responding on the cocaine-paired lever at low doses (1.0-3.0 ng/side), whereas the delta-opioid receptor (DOR) agonist DPDPE induced greater responding at higher doses (300-3000 ng/side) that also enhanced inactive lever responding. Using doses of either agonist that induced responding on only the cocaine-paired lever, we found that DAMGO-induced responding was blocked selectively by pretreatment with the MOR antagonist, CTAP, whereas DPDPE-induced responding was selectively blocked by the DOR antagonist, naltrindole. Cocaine-primed reinstatement was blocked by intra-NAc CTAP but not naltrindole, indicating a role for endogenous MOR-acting peptides in cocaine-induced reinstatement of cocaine-seeking behavior. In this regard, intra-NAc infusions of beta-endorphin (100-1000 ng/side) induced marked cocaine-seeking behavior, an effect blocked by intra-NAc pretreatment with the MOR but not DOR antagonist. Conversely, cocaine seeking elicited by the enkephalinase inhibitor thiorphan (1-10 microg/side) was blocked by naltrindole but not CTAP. MOR stimulation in more dorsal caudate-putamen sites was ineffective, whereas DPDPE infusions induced cocaine seeking. Together, these findings establish distinct roles for MOR and DOR in cocaine relapse and suggest that NAc MOR could be an important therapeutic target to neutralize the effects of endogenous beta-endorphin release on cocaine relapse.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; beta-Endorphin; Cocaine; Cocaine-Related Disorders; Disease Models, Animal; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Male; Narcotic Antagonists; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Recurrence; Reward; Self Administration; Substance Withdrawal Syndrome

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures--the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 microl)--a selective mu-receptor agonist--or nor-binaltorphimine (BNI; 2.5 and 5 microg/0.2 microl)--a selective kappa-receptor antagonist with tardive action--on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region.

Topics: Analgesics, Opioid; Animals; Catheterization; Dose-Response Relationship, Drug; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Escape Reaction; Fear; Freezing Reaction, Cataleptic; Male; Microinjections; Morphine; Naltrexone; Narcotic Antagonists; Rats; Rats, Wistar; Receptors, Opioid, kappa; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Tectum Mesencephali

A single nucleotide polymorphism (SNP) in the human mu-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in a variety of drug addiction and pain sensitivity phenotypes; however, the extent of these adaptations and the mechanisms underlying these associations remain elusive. To clarify the functional mechanisms linking the OPRM1 A118G SNP to addiction and analgesia phenotypes, we derived a mouse model possessing the equivalent nucleotide/amino acid substitution in the Oprm1 gene. Mice harboring this SNP (A112G) demonstrated several phenotypic similarities to humans carrying the A118G SNP, including reduced mRNA expression and morphine-mediated antinociception. We found additional phenotypes associated with this SNP including significant reductions of receptor protein levels, morphine-mediated hyperactivity, and the development of locomotor sensitization in mice harboring the G112 allele. In addition, we found sex-specific reductions in the rewarding properties of morphine and the aversive components of naloxone-precipitated morphine withdrawal. Further cross-species analysis will allow us to investigate mechanisms and adaptations present in humans carrying this SNP.

Topics: Analgesics, Opioid; Animals; Base Sequence; Binding, Competitive; Conditioning, Operant; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Gene Expression; Gene Frequency; Genotype; Humans; Male; Mice; Mice, Inbred C57BL; Models, Animal; Morphine; Motor Activity; Pain; Pain Measurement; Polymorphism, Single Nucleotide; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sex Factors; Substance Withdrawal Syndrome

mu-Opioid receptor (MOR) agonists represent the gold standard for the treatment of severe pain but may paradoxically also enhance pain sensitivity, that is, lead to opioid-induced hyperalgesia (OIH). We show that abrupt withdrawal from MOR agonists induces long-term potentiation (LTP) at the first synapse in pain pathways. Induction of opioid withdrawal LTP requires postsynaptic activation of heterotrimeric guanine nucleotide-binding proteins and N-methyl-d-aspartate receptors and a rise of postsynaptic calcium concentrations. In contrast, the acute depression by opioids is induced presynaptically at these synapses. Withdrawal LTP can be prevented by tapered withdrawal and shares pharmacology and signal transduction pathways with OIH. These findings provide a previously unrecognized target to selectively combat pro-nociceptive effects of opioids without compromising opioid analgesia.

Topics: Analgesics, Opioid; Animals; Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; GTP-Binding Proteins; Hyperalgesia; Long-Term Potentiation; Male; Nerve Fibers, Unmyelinated; Patch-Clamp Techniques; Piperidines; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu; Remifentanil; Signal Transduction; Substance Withdrawal Syndrome; Synapses

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Cells, Cultured; Conditioning, Operant; Corpus Striatum; Cyclic AMP; Embryo, Mammalian; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme-Linked Immunosorbent Assay; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Morphine; Motor Activity; Nerve Tissue Proteins; Neurons; Protein Binding; Rats; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Transfection

Although the expression of the morphine (MOR) withdrawal syndrome is more marked in male mice than in females, we have demonstrated that the GABAB agonist baclofen (BAC) is able to attenuate MOR withdrawal signs in either sex. In order to extend these previous observations, the aim of the present study was to evaluate the mu-opioid receptor labeling in various brain areas in mice of either sex, during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice were rendered dependent by intraperitonial (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the 10th day, dependent animals received naloxone (NAL; 6 mg/kg, i.p.) 60 min after MOR, and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL. Thirty minutes after NAL, mice were sacrificed and autoradiography with [3H]-[D-Ala2, N-Me-Phe4, -glycol5] enkephalin (DAMGO) was carried out on mice brains at five different anatomical levels. Autoradiographic mapping showed a significant increase of mu-opioid receptor labeling during MOR withdrawal in nucleus accumbens core (NAcC), caudate putamen (CPu), mediodorsal thalamic nucleus (MDTh), basolateral and basomedial amygdala, and ventral tegmental area vs. respective control groups in male mice. In contrast, opiate receptor labeling was not significantly modified in any of the brain areas studied in withdrawn females. BAC reestablished mu-opioid receptor binding sites during MOR withdrawal only in NAcC of males, and a similar tendency was observed in CPu and MDTh, even when it was not statistically significant. The sexual dimorphism observed in the present study confirms previous reports indicating a greater sensitivity of males in response to MOR pharmacological properties. The present results suggest that the effect of BAC in preventing the expression of MOR withdrawal signs could be related with the ability of BAC to reestablish the mu-opioid receptor labeling in certain brain areas.

Topics: Aging; Animals; Autoradiography; Baclofen; Binding Sites; Brain; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; GABA Agonists; Male; Mice; Morphine; Morphine Dependence; Narcotic Antagonists; Narcotics; Receptors, Opioid, mu; Sex Characteristics; Substance Withdrawal Syndrome

There is evidence showing that the opioid and adenosine systems play an important role in cocaine addiction; fewer studies have examined their roles in cocaine withdrawal. To determine whether cocaine and/or chronic withdrawal from cocaine alters the specific components of the opioid and adenosine systems, we carried out quantitative autoradiographic mapping of mu-opioid, A1 and A2A adenosine receptors in the brains of rats treated with an escalating dose "binge" cocaine administration paradigm and of rats chronically withdrawn from cocaine. Male Fischer rats were injected with saline or cocaine (15 x 3 mg/kg/day for 4 days, 20 x 3 mg/kg/day for 4 days, 25 x 3 mg/kg/day for 4 days and 30 x 3 mg/kg/day for 2 days) at 1-h intervals for 14 days. Similarly treated rats were withdrawn from that paradigm for 14 days. A significant increase in [(3)H]DAMGO binding to mu-receptors was detected in the frontal and cingulate cortex, as well as in the caudate putamen, of long-term withdrawn rats after an escalating dose "binge" cocaine administration paradigm and in chronic cocaine-treated rats. No significant cocaine-induced change was found in A1 or A2A receptor binding in any region analyzed. These results reconfirm that mu-opioid (MOP) receptors undergo upregulation in response to chronic escalating dose "binge" cocaine administration. This upregulation was shown for the first time to persist at least 14 days into withdrawal after escalating "binge" cocaine.

Topics: Adenosine; Analgesics, Opioid; Animals; Autoradiography; Behavior, Animal; Brain; Brain Mapping; Cocaine; Dopamine Uptake Inhibitors; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Phenethylamines; Protein Binding; Purinergic P1 Receptor Antagonists; Rats; Rats, Inbred F344; Receptors, Opioid, mu; Receptors, Purinergic P1; Substance Withdrawal Syndrome; Tritium; Up-Regulation; Xanthines

Opiates produce analgesia by activating mu opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. After chronic opiate exposure, an excitatory effect emerges contributing to analgesic tolerance and opioid-induced hyperalgesia. Ultra-low-dose opioid antagonist co-treatment blocks the excitatory effects of opiates in vitro, as well as opioid analgesic tolerance and dependence, as was demonstrated here with ultra-low-dose naloxone combined with morphine. While the molecular mechanism for the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the mu opioid receptor and adenylyl cyclase activation by Gbetagamma have both been suggested. Using CNS regions from rats chronically treated with vehicle, morphine, morphine+ultra-low-dose naloxone or ultra-low-dose naloxone alone, we examined whether altered mu opioid receptor coupling to G proteins or adenylyl cyclase activation by Gbetagamma occurs after chronic opioid treatment. In morphine-naïve rats, mu opioid receptors coupled to Go in striatum and to both Gi and Go in periaqueductal gray and spinal cord. Although chronic morphine decreased Gi/o coupling by mu opioid receptors, a pronounced coupling to Gs emerged coincident with a Gbetagamma interaction with adenylyl cyclase types II and IV. Co-treatment with ultra-low-dose naloxone attenuated both the chronic morphine-induced Gs coupling and the Gbetagamma signaling to adenylyl cyclase, while increasing Gi/o coupling toward or beyond vehicle control levels. These findings provide a molecular mechanism underpinning opioid tolerance and dependence and their attenuation by ultra-low-dose opioid antagonists.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Blotting, Western; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Immunoprecipitation; Isoenzymes; Male; Naloxone; Narcotic Antagonists; Opioid-Related Disorders; Pain Threshold; Protein Binding; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.

Topics: Analysis of Variance; Animals; Autoradiography; Behavior, Animal; Binding Sites; Brain; Densitometry; Diarrhea; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Knockout; Morphine; Motor Activity; Naloxone; Narcotic Antagonists; Raclopride; Receptor, Adenosine A2A; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Sulfur Isotopes; Tremor; Tritium; Urine; Weight Loss

We have previously shown that the GABA(B) agonist baclofen (BAC) prevents the expression of morphine (MOR) withdrawal syndrome in male as well as female mice. In addition, we have demonstrated that BAC reestablishes the dopamine levels modified by MOR withdrawal syndrome in male mice. The aim of the present study was to evaluate the micro-opioid receptor binding parameters in striatum and frontal cortex of male and female mice during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice of either sex were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the tenth day, dependent animals received naloxone (NAL) (6 mg/kg, i.p.) 60 min after the last dose of MOR and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL injection. Thirty min after NAL or saline injection mice were sacrificed, brains were collected, and the striatum and frontal cortex were dissected in order to perform binding studies with [(3)H][DAMGO]. The density of micro-opioid receptor increased significantly during MOR withdrawal in male and female striatum as well as in male cortex. In addition, in both brain areas the B(max) was higher in male than in female mice during MOR withdrawal. Finally, BAC pretreatment of MOR withdrawn mice reestablished the levels of micro-opioid receptor by significantly decreasing the B(max) in either sex. In conclusion, although there were sex differences in the micro-opioid receptor density during MOR withdrawal syndrome, BAC was able to reestablish the changes in binding parameters induced by the NAL-precipitated withdrawal in female and male mice.

Topics: Animals; Baclofen; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; GABA Agonists; Male; Mice; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Receptors, Opioid, mu; Sex Factors; Substance Withdrawal Syndrome

The dermorphin-derived tetrapeptide Tyr-D-Arg(2)-Phe-Sar(4) (TAPS) was tested for its ability to induce tolerance, cross-tolerance, withdrawal and its substitution properties in rats subjected to chronic intracerebroventricular (i.c.v.) infusions of mu-opiate receptor agonists. Tolerance and cross-tolerance were assessed by quantification of the thermally induced tail-flick response. Chronic intracerebroventricular infusion of TAPS resulted in antinociception at almost 1000-fold lower doses compared to morphine sulphate and [D-Ala(2), MePhe(4)Gly(ol)(5)]enkephalin (DAMGO). Tolerance to the antinociceptive effect of TAPS developed similar to DAMGO and morphine sulphate. Cross-tolerance to intracerebroventricular bolus injections of DAMGO, but not of TAPS, was evident in rats rendered tolerant to morphine sulphate and TAPS. Naloxone-induced withdrawal was equally pronounced in animals treated with morphine sulphate, DAMGO or TAPS. TAPS substituted for morphine sulphate and vice versa regarding the withdrawal syndrome in a cross-over experimental design. In contrast to DAMGO, TAPS retains its antinociceptive effect following bolus administration in rats rendered tolerant to mu-opioid receptor agonists.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; In Vitro Techniques; Infusion Pumps; Injections, Intraventricular; Male; Morphine; Naloxone; Oligopeptides; Pain; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Substance-Related Disorders; Time Factors

We used in vivo microdialysis in awake rats to test the hypothesis that intravenous morphine increases serotonin (5-HT) release within the rostral ventromedial medulla (RVM). We also injected morphine into various sites along the rostrocaudal extent of the periaqueductal gray (PAG), and examined the extent of its diffusion to the RVM. Intravenous morphine (3.0 mg/kg) produced thermal antinociception and increased RVM dialysate 5-HT, 5-hydroxyindole acetic acid (5-HIAA), and homovanillic acid (HVA) in a naloxone-reversible manner. As neither PAG microinjection of morphine (5 micro g/0.5 micro L) nor RVM administration of fentanyl or d-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased RVM 5-HT, we were unable to determine the precise site of action of morphine. Surprisingly, peak morphine levels in the RVM were higher after microinjection into the caudal PAG as compared to either intravenous injection or microinjection into more rostral sites within the PAG. Naloxone-precipitated withdrawal in morphine-tolerant rats not only increased extracellular 5-HT in the RVM, but also dopamine (DA) and HVA. We conclude that substantial amounts of morphine diffuse from the PAG to the RVM, and speculate that opioid receptor interactions at multiple brain sites mediate the analgesic effects of PAG morphine. Further studies will be required to elucidate the contribution of 5-HT and DA release in the RVM to opioid analgesia and opioid withdrawal.

Topics: Analgesics, Opioid; Animals; Drug Administration Routes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fentanyl; Homovanillic Acid; Hydroxyindoleacetic Acid; Male; Medulla Oblongata; Microdialysis; Microinjections; Morphine; Naloxone; Narcotic Antagonists; Pain Measurement; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Serotonin; Substance Withdrawal Syndrome; Wakefulness

In the present work the effect of papaverine, a non specific smooth muscle relaxant, was investigated on the naloxone-precipitated withdrawal contracture of the acute morphine-dependent guinea-pig ileum in vitro. Furthermore, the effect of papaverine was also considered on DAGO (highly selective mu -agonist) and U50-488H (highly selective k-agonist) withdrawal to test whether the possible interaction of papaverine on opioid withdrawal involves mu - and/or k-opioid receptors. Following a 4 min in vitro exposure to opioid agonist, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. Papaverine treatment (1 x 10(-7) - 5 x 10(-7) - 1 x 10(-6) M) before or after the opioid agonists was able of both preventing and reversing the naloxone-induced contracture after exposure to mu (morphine and DAGO) or k (U50-488H) opiate agonists in a concentration-dependent fascion. Both acetylcholine response and electrical stimulation were not affected by papaverine treatment whereas the final opiate withdrawal was still reduced. The results of the present study indicate that papaverine was able to produce significative influence on the opiate withdrawal in vitro and papaverine was able to exert its effect both at mu and k opioid agonists.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acute Disease; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; Male; Morphine; Muscle Contraction; Muscle, Smooth; Narcotic Antagonists; Papaverine; Parasympatholytics; Phytotherapy; Plants, Medicinal; Receptors, Opioid; Substance Withdrawal Syndrome

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

Topics: Aging; Animals; Avoidance Learning; Brain; Colforsin; Cyclic AMP; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Intermediate Filament Proteins; Locus Coeruleus; Mice; Mice, Knockout; Mice, Transgenic; Morphine; Morphine Dependence; Nerve Tissue Proteins; Nestin; Neurons; Neurotrophin 3; Signal Transduction; Substance Withdrawal Syndrome; Tyrosine 3-Monooxygenase

The nucleus accumbens, as part of the mesolimbic dopaminergic reward pathway, mediates both addiction to and withdrawal from substances of abuse. In addition, activity of substances of abuse such as opioids in the nucleus accumbens has been implicated in pain modulation. Because nucleus accumbens nicotinic receptors are important in nicotine addiction and because nicotinic activity can interact with opioid action, we investigated the contribution of nucleus accumbens nicotinic receptors to opioid-mediated analgesia/antinociception. The response of the nociceptive jaw-opening reflex to opioids was studied in the rat, both before and during chronic nicotine exposure. In nicotine-naive rats, intra-accumbens injection of the nicotinic receptor antagonist mecamylamine blocked antinociception produced by either systemic morphine, intra-accumbens co-administration of a mu- and a delta-opioid receptor agonist, or noxious stimulation (i.e., subdermal capsaicin in the hindpaw); intra-accumbens mecamylamine alone had no effect. The antinociceptive effect of either morphine or noxious stimulation was unchanged during nicotine tolerance; however, intra-accumbens mecamylamine lost its ability to block antinociception produced by either treatment. Intra-accumbens mecamylamine by itself precipitated significant hyperalgesia in nicotine-tolerant rats which could be suppressed by noxious stimulation as well as by morphine. These results indicate that nucleus accumbens nicotinic receptors play an important role in both opioid- and noxious stimulus-induced antinociception in nicotine-naive rats. This role was attenuated in the nicotine-dependent state. The suppression of withdrawal hyperalgesia by noxious stimulation suggests that pain can ameliorate the symptoms of withdrawal, thus suggesting a possible mechanism for pain-seeking behavior.

Topics: Analgesia; Animals; Capsaicin; Dose-Response Relationship, Drug; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hyperalgesia; Male; Mecamylamine; Morphine; Narcotics; Nicotine; Nicotinic Antagonists; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Substance Withdrawal Syndrome; Tobacco Use Disorder

Withdrawal from repeated exposure to morphine causes a long-lasting increase in the reactivity of nucleus accumbens nerve terminals towards excitation. The resulting increase in action potential-induced exocytotic release of neurotransmitters, associated with behavioral sensitization, is thought to contribute to its addictive properties. We recently showed that activation of N-methyl-D-aspartate (NMDA) as well as dopamine (DA) D1 receptors in rat striatum causes tetrodotoxin-insensitive transporter-dependent GABA release. Since sustained changes in extracellular GABA levels may play a role in drug-induced neuronal hyperresponsiveness, we examined the acute and long-lasting effect of morphine on this nonvesicular GABA release in rat nucleus accumbens slices. The present study shows that morphine, through activation of mu-opioid receptors, reduces nonvesicular NMDA-induced [(3)H]GABA release in superfused nucleus accumbens slices. Moreover, prior repeated morphine treatment of rats (10 mg/kg, sc, 14 days) caused a reduction in NMDA-stimulated [(3)H]GABA release in vitro until at least 3 weeks after morphine withdrawal. This persistent neuroadaptive effect was not observed studying dopamine D1 receptor-mediated [(3)H]GABA release in nucleus accumbens slices. Moreover, this phenomenon appeared to be absent in slices of the caudate putamen. Interestingly, even a single exposure of rats to morphine (>2 mg/kg) caused a long-lasting inhibition of NMDA-induced release of GABA in nucleus accumbens slices. These data suggest that a reduction in nonvesicular GABA release within the nucleus accumbens, by enhancing the excitability of input and output neurons of this brain region, may contribute to the acute and persistently enhanced exocytotic release of neurotransmitters from nucleus accumbens neurons in morphine-exposed rats.

Topics: Analgesics; Animals; Benzeneacetamides; Carrier Proteins; Dopamine; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Male; Morphine; Morphine Dependence; N-Methylaspartate; Naloxone; Neural Inhibition; Neurons; Nucleus Accumbens; Organ Culture Techniques; Pyrrolidines; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Tritium

Major adaptations after chronic exposure to morphine include an up-regulation of the adenosine 3',5'-monophosphate pathway. Acute opioids, via mu-opioid receptors, disinhibit midbrain serotonergic neurons by suppressing inhibitory GABAergic transmission in the dorsal raphe nucleus and adjacent periaqueductal gray. This study examined whether chronic morphine induces a compensatory increase in GABA inputs to 5-hydroxytryptamine neurons and whether this was associated with an up-regulation of the adenosine 3',5'-monophosphate pathway. The firing rate of serotonergic neurons was reduced in brain slices from morphine-dependent rats, an effect reversed by the GABA(A) antagonist bicuculline. The reduction in firing rate was accompanied by an increased frequency of spontaneous GABAergic inhibitory postsynaptic currents, indicating increased GABA tone in the slice. The increase in GABA tone in brain slices from dependent rats was associated with increased induction of inhibitory postsynaptic currents by the adenylyl cyclase activator forskolin, suggesting an up-regulation of the adenosine 3',5'-monophosphate pathway. Indeed, chronic morphine increased levels of adenylyl cyclase VIII (but not of adenylyl cyclase I, III or V) immunoreactivity in the dorsal raphe nucleus area. Two adenosine 3',5'-monophosphate-mediated mechanisms for the increase in GABA tone were discerned. The first, which predominated when impulse-flow was blocked by tetrodotoxin, involves protein kinase A since it was sensitive to protein kinase A inhibitors. The second, seen when impulse-flow was intact (i.e. absence of tetrodotoxin), was insensitive to protein kinase A inhibitors but was suppressed by ZD7288, a blocker of hyperpolarizing-activated Ih channels which are directly activated by adenosine 3',5'-monophosphate. We conclude that chronic morphine induces an up-regulation of the adenosine 3',5'-monophosphate pathway in GABAergic inputs to serotonergic cells, resulting in an increase in spontaneous and impulse-flow dependent GABA release. These changes would lead to an increase in GABA tone and subsequently to the reported decrease in serotonergic activity during opiate withdrawal.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cardiovascular Agents; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Isoquinolines; Male; Membrane Potentials; Morphine; N-Methylaspartate; Neural Inhibition; Periaqueductal Gray; Phenylephrine; Pyrimidines; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Serotonin; Substance Withdrawal Syndrome; Sulfonamides; Sympathomimetics; Tetrazoles; Tetrodotoxin; Thionucleotides

Previous studies have shown that chronic ethanol influences the density of central mu-opioid receptors and serotonin(1A) (5-hydroxytryptamine(1A)) receptors. To determine whether the functional coupling of these two receptors to G proteins in the rat brain, particularly in mesocorticolimbic regions, is affected by ethanol, receptor-mediated [(35)S]guanosine-5'-O-(3-thio)-triphosphate ([(35)S]GTPgammaS) binding stimulated by [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO) or L694,247 was used. By quantitative autoradiography, receptor-mediated [(35)S]GTPgammaS binding activated by the two agonists was mapped throughout brain sections at the level of the nucleus accumbens and hippocampus from groups of alcohol-preferring Fawn-Hooded (FH) rats after different ethanol consumption paradigms. Significant DAMGO (mu-opioid receptor agonist)-stimulated binding of [(35)S]GTPgammaS was obtained in the striatum, nucleus accumbens, and lateral septum, whereas L694,247 (5-hydroxytryptamine(1A/1B/1D) receptor agonist)-stimulated binding of [(35)S]GTPgammaS was observed in the lateral septum, amygdala, and cingulate cortex. Chronic ethanol self-administration significantly reduced DAMGO-stimulated [(35)S]GTPgammaS binding in the nucleus accumbens (-19%), lateral septum (-15%), and striatum (-23%), which recovered toward control levels after ethanol withdrawal. However, chronic ethanol, as well as ethanol withdrawal, failed to produce any significant alteration in L694,247-stimulated [(35)S]GTPgammaS binding in all tested brain regions. The region-specific and receptor-specific alteration of agonist-stimulated [(35)S]GTPgammaS binding suggests that the change of functional coupling of mu-opioid receptors to G proteins induced by chronic ethanol drinking may have a pathophysiological role in the consequences of ethanol consumption.

Topics: Alcohol Drinking; Animals; Autoradiography; Brain Chemistry; Central Nervous System Depressants; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ethanol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Protein Binding; Rats; Rats, Inbred Strains; Receptors, Opioid, mu; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Substance Withdrawal Syndrome

Numerous reports support evidence that dopaminergic mesolimbic pathways interact with opioid systems to influence the reinforcing properties of cocaine. Withdrawal from chronic administration of cocaine in rats causes an upregulation of mesocorticolimbic mu-opioid receptors during early stages, but information about prolonged cocaine abstinence is lacking. We addressed this issue by treating rats with cocaine or saline (control) intermittently (1 mg/kg, i.v., every 12 min for 2 h daily) for 10 days followed by a 10- or 20-day withdrawal period. The animals were then decapitated and the brains removed for quantitative in vitro autoradiographic analysis of 14 brain regions with (125)I-DAMGO. A separate group of animals received two consecutive cycles of the 10-day cocaine/10-day withdrawal regimen. Only the group that participated in the two consecutive cycles showed a significant effect of treatment: downregulation of mu-opiate receptors in limbic cortical layer 3 (17% lower than saline-treated controls, P = 0.03), the core of the nucleus accumbens (16% decrease, P = 0.05), and the nucleus of the diagonal band (18% decrease, P = 0.05). The mu-receptor may manifest, as do other neural markers (e.g., dopamine transporter, dopamine efflux), a biphasic temporal pattern with upregulation during early phases of cocaine withdrawal but a downregulation at later times.

Topics: Analgesics, Opioid; Animals; Autoradiography; Cocaine; Cocaine-Related Disorders; Dopamine Uptake Inhibitors; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Iodine Radioisotopes; Limbic System; Male; Nucleus Accumbens; Rats; Rats, Inbred Lew; Receptors, Opioid, mu; Septal Nuclei; Substance Withdrawal Syndrome

Rats, for 8 weeks consuming the mu-opioid agonist etonitazene (forced and free choice conditions yielding high and low drug-consumers), were sacrificed after 2 days or 6 weeks lasting drug deprivation. Binding characteristics of membranes from the parieto-occipital cortex of these four groups were compared with those of drug-naive controls. In all five groups, 1 microM of the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) increased the guanosine-5'-O([35S]3'thio)triphosphate ([35S]GTPgammaS) binding activity on guanine nucleotide-binding (G) proteins, and 500 nM of GTPgammaS decreased the [3H]DAMGO binding affinity. During acute withdrawal, both opioid consuming groups displayed a higher maximum efficacy (Emax) in basal [35S]GTPgammaS binding (34 and 31%, each P < 0.01), but only the forced group showed a 58% higher net DAMGO-stimulated binding density Bmax (P < 0.01) and 53% more activated G proteins per mu-opioid receptor (P < 0.05). In the presence of GTPgammaS both groups revealed a higher affinity in [3H]DAMGO binding (each 25%, P < 0.01). The long-term drug-deprived groups displayed no differences in their binding characteristics.

Topics: Animals; Benzimidazoles; Brain; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Narcotics; Rats; Rats, Wistar; Receptors, Opioid, mu; Self Administration; Signal Transduction; Substance Withdrawal Syndrome; Substance-Related Disorders

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed mu-opioid receptors in rats treated with morphine and diazepam for 5 days by using [3H]-DAMGO for binding studies. Male Sprague-Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of mu-receptor (Bmax increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of mu-receptors in spinal cord of morphine treated rats. In the cortex, Bmax was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The Kd of mu-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the Kd of mu-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS mu-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of mu-opioid receptors to mu-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Brain Chemistry; Central Nervous System; Diazepam; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hypnotics and Sedatives; Male; Morphine; Naloxone; Narcotic Antagonists; Nociceptors; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Tritium

The present study investigated the possible role of nitric oxide (NO) in the development of the withdrawal contractures of guinea pig isolated ileum after acute activation of mu- and kappa-opioid receptors. After a 4-min in vitro exposure to morphine (mu-opioid receptor preferring, but not selective, agonist), [D-Ala2-N-methyl-Phe4-Gly5-ol-]enkephalin (DAMGO; highly selective mu-opioid receptor agonist), or trans(+/-)-3,4-dichloro-N-methyl-N-2(1-pyrrolidynyl)cyclohexyl-ben zeneacetamide (U50-488H; highly selective kappa-opioid receptor agonist), the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. L-N(G)-nitro arginine methyl ester (3-300 microM) injected 10 min before the opioid receptor agonists was able dose dependently to reduce the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonists whereas D-N(G)-nitro arginine methyl ester at the same concentrations did not affect it. The inhibitory effect of L-N(G)-nitro arginine methyl ester on morphine, DAMGO and U50-488H withdrawal was dose dependently reversed by L-arginine (3-300 microM) but not by D-arginine. Finally, glyceryl trinitrate on its own (3-300 microM) significantly increased the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonist and it was also able to reverse the inhibition of opioid withdrawal caused by L-N(G)-nitro arginine methyl ester. These results provide evidence that NO has a role in the development of opioid withdrawal and that mu- or kappa-opioid receptors are involved.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acetylcholine; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Guinea Pigs; Ileum; In Vitro Techniques; Male; Morphine; Muscle Contraction; Naloxone; Narcotic Antagonists; Narcotics; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroglycerin; Receptors, Opioid, kappa; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Vasodilator Agents

We examined the interactions among three classes of peripherally-acting antinociceptive agents (mu-opioid, alpha 2-adrenergic, and A1-adenosine) in the development of tolerance and dependence to their antinociceptive effects. Antinociception was determined by assessing the degree of inhibition of prostaglandin E2 (PGE2)-induced mechanical hyperalgesia, using the Randall-Selitto paw-withdrawal test. Tolerance developed within 4 hr to the antinociceptive effect of the alpha 2-adrenergic agonist clonidine; dependence also occurred at that time, demonstrated as a withdrawal hyperalgesia that was precipitated by the alpha 2-receptor antagonist yohimbine. These findings are similar to those reported previously for tolerance and dependence to mu and A1 peripheral antinociception (Aley et al., 1995). Furthermore, cross-tolerance and cross-withdrawal between mu, A1, and alpha 2 agonists occurred. The observations of cross-tolerance and cross-withdrawal suggest that all three receptors are located on the same primary afferent nociceptors. In addition, the observations suggest that the mechanisms of tolerance and dependence to the antinociceptive effects of mu, A1, and alpha 2 are mediated by a common mechanism. Although any of the agonists administered alone produce antinociception, we found that mu, A1, and alpha 2 receptors may not act independently to produce antinociception, but rather may require the physical presence of the other receptors to produce antinociception by any one agonist. This was suggested by the finding that clonidine (alpha 2-agonist) antinociception was blocked not only by yohimbine (alpha 2-antagonist) but also by PACPX (A1-antagonist) and by naloxone (mu-antagonist), and that DAMGO (mu-agonist) antinociception and CPA (A1-agonist) antinociception were blocked not only by naloxone (mu-antagonist) and PACPX (A1-antagonist), respectively, but also by yohimbine (alpha 2-antagonist). This cross-antagonism of antinociception occurred at the ID50 dose for each antagonist at its homologous receptor. To test the hypothesis that the physical presence of mu-opioid receptor is required not only for mu antinociception but also for alpha 2 antinociception, antisense oligodeoxynucleotides (ODNs) for the mu-opioid and alpha 2C-adrenergic receptors were administered intrathecally to reduce the expression of these receptors on primary afferent neurons. These studies demonstrated that mu-opioid ODN administration decreased not only mu-opioid but also alpha 2

Topics: Adenosine; Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Analgesics; Analgesics, Opioid; Animals; Clonidine; Dinoprostone; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hyperalgesia; Male; Models, Biological; Naloxone; Narcotic Antagonists; Oligonucleotides, Antisense; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Receptors, Opioid, mu; Receptors, Purinergic P1; Second Messenger Systems; Substance Withdrawal Syndrome; Substance-Related Disorders; Xanthines; Yohimbine

Chronic opioid regulation of stimulatory beta-2 adrenoceptor (beta-2 AR) signaling was investigated in human mammary epidermoid carcinoma A431 cells stably expressing the cloned rat mu opioid receptor. In the cell clone used (A431/mu 13; Bmax = 302.9 +/- 46 fmol/mg membrane protein), the addition of morphine acutely attenuated basal as well as (-)-isoproterenol-stimulated cAMP accumulation. Prolonged exposure of the cells to morphine (10 microM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/mu 13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal falls to precipitate a cAMP overshoot in this cell system. Sensitization of stimulatory AC signaling by chronic morphine develops in a time- and dose-dependent manner and is blocked by both naloxone and pertussis toxin. Investigation into the mechanism leading to up-regulation of AC revealed a 40% increase in the number of beta-2 ARs as assessed by [125I]-cyanopindolol binding experiments. No additional quantitative changes were found for stimulatory G proteins and the effector enzyme itself. Sensitization of AC appears to be mediated solely by the increase in beta-2 AR numbers, because (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol hydrochloride, which acts as an "inverse agonist" at the beta-2 AR, completely reversed elevated basal AC activities, and because the ratio between functional active beta-2 ARs and stimulatory G proteins remained unchanged. In conclusion, chronic exposure of clonal A431/ mu13 cells to morphine increases the capacity of stimulatory AC signaling by up-regulating beta-2 AR number. These results demonstrate participation of stimulatory receptor systems in the cellular mechanisms underlying opioid dependence.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cells, Cultured; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Isoproterenol; Morphine; Rats; Receptors, Adrenergic, beta-2; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Transfection; Up-Regulation

Previous radioligand-binding studies have reported conflicting results concerning the effect of chronic morphine administration on the regulation of mu-opioid receptor (MOR) density. On the other hand, chronic administration of an opioid antagonist, such as naltrexone, has been shown to increase the density of the MOR. In order to determine if the changes in the MOR are associated with alterations in receptor mRNA levels, we investigated MOR gene expression following chronic treatment with morphine and/or naltrexone. MOR mRNA levels, determined by the ribonuclease protection assay (RPA), were unchanged with respect to control during chronic morphine treatment and morphine withdrawal in each of the analysed brain areas. Furthermore, chronic administration of naltrexone did not result in changes of MOR mRNA levels in rat striatum of naive and morphine-dependent rats, suggesting that the up-regulation of the MOR density, at least in this tissue, is not regulated at transcriptional level.

Topics: Animals; Brain; Corpus Striatum; DNA Primers; Drug Administration Schedule; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Morphine; Naltrexone; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; RNA, Messenger; Substance Withdrawal Syndrome

1. The effects exerted by D1 and D2 dopamine agonists and antagonists on the acute opiate withdrawal induced by mu- and kappa-receptor agonists were investigated in vitro. 2. Following a 4 min in vitro exposure to morphine (moderately selective mu-agonist), [D-Ala2, Me-Phe4, Gly-ol5]enkephalin (DAMGO, highly selective mu-agonist) or U-50488H (highly selective kappa-agonist) the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. 3. The non-selective dopamine receptor antagonist haloperidol when added before or after the opioid agonists, was able dose-dependently to prevent or to reverse the naloxone-induced contracture after exposure to mu- (morphine and DAMGO) and kappa- (U-50488H) opioid agonists. The non-selective dopamine receptor agonist, apomorphine, was able to exert the same effects only at the highest concentration used. 4. The selective D2 dopamine receptor antagonist, sulpiride, was also able to reduce dose-dependently both mu- and kappa-opioid withdrawal, whereas the D1-receptor selective antagonist SCH 23390 did not affect either mu- or kappa-opioid withdrawal. 5. Bromocriptine, a D2 selective dopamine receptor agonist was able to increase significantly, and in a concentration-dependent manner, the naloxone-induced contracture by mu- and kappa-opioid agonists, whereas SKF 38393, a D1 selective dopamine receptor agonist, increased only the withdrawal after morphine or U50-488H. 6. Our data indicate that both D1 and D2 dopamine agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the dopaminergic system and opioid withdrawal at both the mu- and kappa-receptor level. 7. Furthermore, the ability of sulpiride to block strongly opiate withdrawal when compared to SCH 23390, as well as the effect of bromocriptine to increase opiate withdrawal suggest that D2 dopamine receptors may be primarily involved in the control of opiate withdrawal.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Apomorphine; Dopamine Agents; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Haloperidol; Ileum; In Vitro Techniques; Male; Morphine; Muscle, Smooth; Pyrrolidines; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Opioid, kappa; Receptors, Opioid, mu; Substance Withdrawal Syndrome

The effect of dexamethasone on acute opiate withdrawal induced by mu, kappa and delta receptor agonists was investigated in vitro. After a 4-min in vitro exposure to morphine (less selective mu agonist), D-Ala2-N-methyl-Phe4-Gly5-ol)-enkephalin (DAGO; highly selective mu agonist) and trans(+/-)-3,4-dichloro-N-methyl-N-[2(1-pyrrolidynyl)cyclohexyl]- benzeneacetamide (U50-488H; highly selective kappa agonist) a strong contracture of guinea pig isolated ileum was observed after the addition of naloxone. This effect was also observed when rabbit isolated jejunum was pretreated with deltorphin (highly selective delta agonist). Dexamethasone treatment before or after the opioid agonists tested was capable of both preventing and reverting the naloxone-induced contracture after exposure to mu opiate agonists morphine and DAGO in a concentration- and time-dependent fashion. Also, the steroid reduced naloxone-induced contracture after the exposure to U50-488H only when injected before the kappa opiate agonist. Finally, it did not affect the naloxone contracture after exposure to deltorphin. Pretreatment with RU-38486, a glucocorticoid receptor antagonist, inhibited dexamethasone antagonism on responses to both mu and kappa agonists, whereas pretreatment with cycloheximide, a protein synthesis inhibitor, blocked only the antagonistic effects of dexamethasone on responses to the mu opioid agonists. Overall, these data indicate that dexamethasone induces significant effects on mu-mediated opiate with-drawal in vitro, which suggest an important functional interaction between corticosteroids and the opioid system primarily at the mu receptor level. The ability of RU-38486 and cycloheximide to block dexamethasone effects indicates that the steroid interference on mu-mediated withdrawal involves a protein synthesis-dependent mechanism via glucocorticoid receptor.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Cycloheximide; Dexamethasone; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; In Vitro Techniques; Male; Mifepristone; Muscle Contraction; Naloxone; Opioid-Related Disorders; Pyrrolidines; Rabbits; Substance Withdrawal Syndrome

1. Morphine produces a plethora of pharmacological effects and its chronic administration induces several side-effects. The cellular mechanisms by which opiates induce these side-effects are not fully understood. Several studies suggest that regulation of adenylyl cyclase activity by opioids and other transmitters plays an important role in the control of neural function. 2. The aim of this study was to evaluate desensitization of mu- and delta- opioid receptors, defined as a reduced ability of opioid agonists to inhibit adenylyl cyclase activity, in four different brain structures known to be involved in opiate drug actions: caudate putamen, nucleus accumbens, thalamus and periaqueductal gray (PAG). Opiate regulation of adenylyl cyclase in these regions has been studied in control and morphine-dependent rats. 3. The chronic morphine treatment used in the present study (subcutaneous administration of 15.4 mg morphine/rat/day for 6 days via osmotic pump) induced significant physical dependence as indicated by naloxone-precipitated withdrawal symptoms. 4. Basal adenylyl cyclase in the four brain regions was not modified by this chronic morphine treatment. In the PAG and the thalamus, a desensitization of mu- and delta-opioid receptors was observed, characterized by a reduced ability of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO; mu), Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; delta) and [D-Ala2]-deltorphin-II (DT-II; delta) to inhibit adenylyl cyclase, activity following chronic morphine treatment. 5. The opioid receptor desensitization in PAG and thalamus appeared to be heterologous since the metabotropic glutamate receptor agonists, L-AP4 and glutamate, and the 5-hydroxytryptamine (5-HT)1A receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), also showed reduced inhibition of adenylyl cyclase activity following chronic morphine treatment. 6. In the nucleus accumbens and the caudate putamen, desensitization of delta-opioid receptor-mediated inhibition without modification of mu-opioid receptor-mediated inhibition was observed. An indirect mechanism probably involving dopaminergic systems is proposed to explain the desensitization of delta-mediated responses and the lack of mu-opioid receptor desensitization after chronic morphine treatment in caudate putamen and nucleus accumbens. 7. These results suggest that adaptive responses occurring during chronic morphine administration are not identical in all opiate-sensitive neural populations.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Adenylyl Cyclases; Aminobutyrates; Analgesics; Animals; Brain; Caudate Nucleus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glutamic Acid; Male; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Nucleus Accumbens; Oligopeptides; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Serotonin Receptor Agonists; Substance Withdrawal Syndrome; Thalamus

Despite tremendous efforts in the search for safe, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Opioids exert their pharmacological actions through three opioid-receptor classes, mu, delta and kappa, whose genes have been cloned. Genetic approaches are now available to delineate the contribution of each receptor in opioid function in vivo. Here we disrupt the mu-opioid-receptor gene in mice by homologous recombination and find that there are no overt behavioural abnormalities or major compensatory changes within the opioid system in these animals. Investigation of the behavioural effects of morphine reveals that a lack of mu receptors abolishes the analgesic effect of morphine, as well as place-preference activity and physical dependence. We observed no behavioural responses related to delta- or kappa-receptor activation with morphine, although these receptors are present and bind opioid ligands. We conclude that the mu-opioid-receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component of the opioid system for morphine action.

Topics: Analgesics; Animals; Behavior, Animal; Cell Line; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gene Deletion; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Morphine Dependence; Narcotics; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reward; Substance Withdrawal Syndrome

The goal of this study was to compare the characteristics of mu- and delta-opioid receptors in the cortex of DBA/2 and C57BL/6 mice, which differ in sensitivity to the long- and short-term effects of morphine. The characteristics of mu-opiate receptors were not different in the cortex of both strains. Both high- and low-affinity binding sites of DSLET, a specific ligand of delta-opiate receptors, were present in the cortex of C57BL/6 mice, whereas the high-affinity binding sites were not found in the cortex of DBA/2 mice. The absence of high-affinity DSLET binding sites, which are similar to the delta 2 type of opioid receptors, may explain the less intensive naloxone-precipitated withdrawal reaction of DBA/2 as compared with C57BL/6 mice.

Topics: Amino Acid Sequence; Analgesics; Animals; Drug Implants; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; Morphine; Morphine Dependence; Naloxone; Narcotics; Receptors, Opioid, delta; Receptors, Opioid, mu; Substance Withdrawal Syndrome

1. The effect of morphine tolerance and abstinence on the binding of [3H]naltrexone to discrete brain regions and spinal cord of the rat was determined. 2. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with six morphine pellets for a 7-day period. Each pellet contained 75 mg of morphine base. Rats implanted with six placebo pellets each served as controls. 3. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to various doses of morphine. 4. The binding characteristics (Bmax or Kd values) of [3H]naltrexone, an opiate receptor antagonist, were determined in various tissues of morphine tolerant and abstinent rats. In the tolerant rats, the pellets were left in place at the time of sacrificing, whereas in the abstinent rats, the pellets were removed 18 hr prior to sacrificing. 5. The binding of [3H]naltrexone to opiate receptors on membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups was determined. 6. [3H]Naltrexone bound to tissue membranes at a single high affinity binding sites. The Bmax values of [3H]naltrexone to bind to opiate receptors on the membranes of amygdala and striatum were increased significantly in morphine tolerant rats when compared to the placebo controls, but the Kd values did not differ. 7. The Bmax and Kd values of [3H]naltrexone did not differ in any other brain region or spinal cord of morphine tolerant rats and their placebo controls. The binding constants of [3H]naltrexone were unaffected in morphine abstinent rats. 8. Previously we had shown that the binding of [3H]D-Ala2, MePhe4, Gly-ol5 enkephalin (DAMGO), a highly specific agonist for mu-opiate receptors was decreased in cortex, pons and medulla and spinal cord of morphine tolerant but not in the abstinent rats. In addition, delta and kappa receptors are unaffected in morphine tolerant and abstinent rats. 9. The results suggest that direction of change, as well as, the brain areas for mu-agonist and -antagonist opiate binding sites are affected differentially in morphine tolerant rats.

Topics: Analgesics; Animals; Brain; Drug Implants; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Membranes; Morphine; Morphine Dependence; Naltrexone; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, mu; Spinal Cord; Substance Withdrawal Syndrome

Bilateral injection of naloxone (3.0-30.0 nmol) into the substantia nigra of morphine-dependent rats produced a withdrawal syndrome consisting of wet-dog shakes, teeth chattering, irritability to touch, diarrhea and hypothermia. Intense wet-dog shakes and grooming were observed after intranigral injection of the mu selective antagonist D-Phe-Cys-Try-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 3.0-30.0 nmol) in morphine-dependent animals. Body temperature after 30.0 nmol CTOP was significantly increased. A significant positive correlation between body temperature and wet-dog shakes was observed in morphine-dependent animals that received CTOP. Intranigral injection of beta-funaltrexamine (beta-FNA, 10.0 nmol), an irreversible mu antagonist, produced no signs of withdrawal in morphine-dependent animals. However, intranigral injection of beta-FNA (1.0-3.0 nmol) suppressed the antinociceptive effect of the mu-selective agonist, D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAGO, 1.0 nmol). The withdrawal syndrome produced by CTOP (10.0 nmol) was not suppressed by the administration of U50,488H (10.0 nmol), a kappa agonist, suggesting that the absence of an effect of beta-FNA was not due to its kappa agonist activity. Neither the delta-selective antagonist, naltrindole (NTI, 10.0 nmol) nor the kappa-selective antagonist, nor-binaltorphimine (nor-BNI, 10.0 nmol) produced withdrawal. Only wet-dog shakes were observed when CTOP, NTI and nor-BNI (5 nmol each) were administered together into the nigra. These studies suggest an involvement of mu receptors in the nigra in the wet-dog shakes and thermoregulatory dysfunction that occur during withdrawal of morphine. However, the subtypes of opioid receptors in the nigra, that mediate the other signs of morphine withdrawal remain obscure.

Topics: Amino Acid Sequence; Analgesics; Animals; Behavior, Animal; Body Temperature; Brain; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Molecular Sequence Data; Morphine; Morphine Dependence; Naloxone; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Stereotaxic Techniques; Substance Withdrawal Syndrome; Substantia Nigra

Topics: Animals; Brain Chemistry; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Ethylketocyclazocine; In Vitro Techniques; Male; Morphine; Morphine Dependence; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord; Substance Withdrawal Syndrome

Topics: Animals; Behavior, Animal; Body Temperature; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hydroxamic Acids; Naloxone; Narcotics; Oligopeptides; Phenylalanine; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Weight Loss

The aim of the present study was to investigate if a physical dependence could be induced by chronic activation of the endogenous enkephalinergic system. We have therefore evaluated naloxone-induced withdrawal syndrome in rats after central infusion during 7 days of comparable antinociceptive doses of RB 38 A ((R,S)HONH-CO-CH2-CH(CH2C6H5)-CONH-CH(CH2C6H5)-COOH), a mixed enkephalin catabolism blocker and of the selective mu, DAGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) and delta, DSTBULET (Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr), opioid agonists. The responses were compared to those induced by RB 38 B ((S,S)HONH-CO-CH2-CH(CH2C6H5)-CONH-CH(CH2C6H5)-COOH), a selective inhibitor of the 24.11 neutral endopeptidase (NEP) 'enkephalinase'. DAGO induced a severe withdrawal syndrome evidenced by a large weight loss, hypothermia, jumping, mastication, teeth chattering, diarrhoea, lacrimation and salivation. In contrast, DSTBULET and RB 38 A produced only a moderate physical dependence. Only two signs were statistically different in these two groups: wet dog shakes and temperature. Chronic i.c.v. administration of DAGO, DSTBULET and RB 38 A produced a time-dependent reduction in analgesia, but 120 h after continuous infusion only RB 38 A was able to still induce a significative antinociceptive effect. The present data suggest that even in the drastic conditions used here long-term complete inhibition of enkephalin catabolism induces a weak tolerance and a moderate physical dependence, similar to that produced by delta opioid agonists. This effect was not observed after chronic selective inhibition of NEP by RB 38 B.

Topics: Animals; Cerebral Ventricles; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hydroxamic Acids; Injections, Intraventricular; Male; Naloxone; Neprilysin; Oligopeptides; Phenylalanine; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Reference Values; Substance Withdrawal Syndrome; Substance-Related Disorders

The continuous intrathecal infusion of morphine (2, 6, 20 nmol/h), sufentanil (0.06, 0.2, 0.6 nmol/h), [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAMGO) (0.1, 0.3, 1.0 nmol/h) or [D-Ala2,D-Leu5]enkephalin (DADLE) (2, 6, 20 nmol/h) in unanesthetized rats produces a dose-dependent increase in hot plate latency 1 day after pump implant followed by a gradual return to baseline values by days 3-4, i.e. tolerance. Rats assessed for opioid dependence after 7 days of intrathecal (i.t.) infusion of opioids show a withdrawal syndrome most readily noted by withdrawal body shakes (WBS) after injection of the opioid antagonist, naloxone (1 mg/kg i.p.). The number of WBS was proportional to the infusion dose of opioid agonist. Although each tolerance-producing agent was infused in one of three log-spaced (low, medium, high) doses, selected to have approximately equal antinociceptive activity across agents, the agents varied in the apparent degree of dependence. Thus, at the highest infusion dose, the average number of WBS observed was greatest for DADLE (32.8), morphine (30.2) and sufentanil (25.0) while animals treated with DAMGO displayed a significantly less degree of opioid dependence (8.7).

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Fentanyl; Injections, Spinal; Male; Morphine; Naloxone; Narcotics; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Substance-Related Disorders; Sufentanil; Time Factors