N-nitro-L-arginine reverses L-arginine induced changes in morphine antinociception and distribution of morphine in brain regions and spinal cord of the mouse

Twice daily injections of L-arginine (200 mg/kg, i.p.), a precursor for nitric oxide (NO), for 4 days decreased morphine antinociception in male Swiss–Webster mice. Chronic treatment with L-arginine also produced significant decreases in morphine levels in midbrain, pons and medulla, hippocampus, corpus striatum and spinal cord of mice following an injection of morphine(10 mg/kg, s.c.) in comparison to vehicle-injected mice. N^G-nitro-L-arginine(L-NNA), an inhibitor of NO synthase(NOS),(5 mg/kg, i.p.) given prior to each injection of L-arginine reversed the effects of the latter on morphine antinociception and decreases in morphine levels in brain regions and spinal cord. Chronic injections of L-NNA alone did not modify either morphine antinociception or morphine distribution in brain regions and spinal cord of mice. These results suggest that decreases in morphine antinociception by chronic treatment with L-arginine is related to the decreases in the entry of morphine in the central sites. The reversal of L-arginine-induced effects by L-NNA suggests that NO-NOS system may be playing a critical role in the regulation of blood-brain barrier to morphine.     

Involvement of opioid and non-opioid mechanisms in footshock-induced enhancement of brain histamine turnover in mice

Naloxone (1-10 mg/kg, i.p.) dose-dependently inhibited the footshock-induced elevation in levels of tele-methylhistamine (t-MH), a predominant metabolite of brain histamine (HA), although this compound had no effect on the HA dynamics in the non-shocked control mice. Footshock significantly enhanced the HA depletion induced by alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase. However, in mice treated with naloxone (5 mg/kg, i.p.) footshock did not significantly facilitate the alpha-fluoromethylhistidine-induced HA depletion. In mice which had been rendered morphine-tolerant following an s.c. implantation of a pellet containing 50 mg of morphine base 3 days before, footshock produced no significant elevation of the t-MH level. The treatment with alpha-methyl-p-tyrosine, p-chlorophenylalanine or atropine had no significant influence on the footshock-induced t-MH elevation. The t-MH elevation was the most marked in the midbrain. In the hypothalamus and pons-medulla oblongata, no significant change in the t-MH level was produced by footshock. These results suggest that footshock increases the HAergic activity in the mouse brain partly through activation of opioid-related mechanisms and that alterations in HA dynamics differ with region of the brain.     

N(2)O stimulates NOS enzyme activity in C57BL/6 but not DBA/2 mice

Exposure to 70% N(2)O produces a prominent antinociception in C57BL/6 mice but not DBA/2 mice. N(2)O exposure also increases conversion of [14C]L-arginine to [14C]L-citrulline in homogenates prepared from whole brains of C57BL/6 mice; there was no such increase in NOS activity in the DBA/2 whole brain. A differential N(2)O effect on brain NOS in these inbred strains might explain why the C57BL/6 but not DBA/2 mice are responsive to N(2)O antinociception.     

Enhancement of morphine-induced analgesia after repeated injections of methylenedioxymethamphetamine

Repeated administration of methylenedioxymethamphetamine (MDMA) to rats results in long-term depletion of serotonin (5-hydroxytryptamine; 5-HT) in several brain regions. Because of the apparent role of 5-HT in morphine-induced antinociception, the present experiment was designed to determine the effects of repeated MDMA injections on morphine-induced analgesia. Rats (n = 48) received 8 s.c. injections (one every 12 h for 4 days) of MDMA (20 mg/kg) or saline (1.0 ml/kg). Two weeks after the last injection, the groups were divided into 4 subgroups that received either saline, or morphine 2.5, 3.55 or 5.0 mg/kg (s.c.). Nociception was assayed before and after saline or morphine administration by the method of tail immersion in warm water (55 degrees C). The day after analgesia testing, the animals were sacrificed, brains and spinal cords removed and 5-HT, norepinephrine (NE) and dopamine (DA) levels in various brain and spinal cord regions were assayed. The analgesic effect of morphine was enhanced in rats that had received repeated MDMA injections. MDMA selectively depleted 5-HT in the cortex, hippocampus, striatum, brainstem and in the cervical portion of spinal cord. However, 5-HT levels were not changed in the thoracic and lumbar segments of the spinal cord. Thus, a functional consequence of repeated MDMA administration in rats was to enhance morphine-induced antinociception in association with reductions in brain and cervical spinal cord 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine and serotonin release in dorsal striatum and nucleus accumbens is differentially modulated by morphine in DBA/2J and C57BL/6J mice

Numerous studies have demonstrated that genetic factors significantly influence opioid ability to induce behavioral modification in mice. This differential sensitivity has been extensively studied, particularly in the DBA/2J and C57BL/6J strains. In the present study, using the "in vivo" microdialysis technique in these strains, we investigated the effect of morphine administration on the extracellular levels of dopamine (DA), serotonin (5-HT), and their metabolites in the nucleus accumbens and dorsal striatum--areas thought to be involved in morphine-induced locomotor hyperactivity. In the nucleus accumbens, morphine (20 mg/kg) significantly increased extracellular levels of DA in both strains. However, in dorsal striatum the morphine-induced increase of extracellular DA was lower in DBA/2J mice than in C57BL/6J. Moreover, morphine significantly stimulated 5-HT and 5-hydroxyindolacetic acid (5-HIAA) release both in nucleus accumbens and dorsal striatum of C57BL/6J mice, whereas it decreased 5-HT release without modifying 5-HIAA levels in DBA/2J mice. These results suggest that the different behavioral and biochemical responses to acute morphine described in these two strains could be mediated by different sensitivity of both the dopaminergic and the serotonergic systems.     

Effect of depletion of spinal cord norepinephrine on morphine-induced antinociception

We studied whether antinociception produced by injection of morphine into the nucleus reticularis paragigantocellularis (NRPG) or by superfusion onto the spinal cord involved norepinephrine (NE)-containing neurons that descend from brainstem into the spinal cord. Spinal cord NE concentrations were depleted with the neurotoxin, 6-hydroxydopamine, and antinociception was measured following morphine injection into NRPG or onto spinal cord. Depletion of cord NE by approximately 90% did not attenuate the antinociceptive effect of either 2 or 10 micrograms of morphine injected intrathecally. In contrast, the depletion did significantly attenuate the antinociceptive effect of 2.5 micrograms morphine injected bilaterally into the NRPG. These results suggest that NE-containing neurons descending from brainstem nuclei into the spinal cord are not important in the analgesia produced by injecting morphine directly onto the spinal cord but may be involved with analgesia produced by morphine injection into the NRPG.     

Functional interference of dexamethasone on some morphine effects: hypothesis for the steroid-opioid interaction

The effect of dexamethasone (DEX) and its interaction with morphine has been studied on transmurally-stimulated guinea-pig ileum preparation, gastrointestinal transit and analgesia. TRANSMURALLY-STIMULATED GUINEA-PIG ILEUM PREPARATION: DEX dose-dependently reduced the contractions of the ileum. Proteic synthesis inhibitors did not modify the inhibition induced by DEX whereas RU-38486, a glucocorticoid antagonist receptor, antagonized completely the inhibitory effect of DEX. GASTROINTESTINAL TRANSIT: DEX was found to antagonize morphine-, atropine- and verapamil-induced constipation. Cycloheximide does not modify the DEX effects. RU-38486 reverses both the inhibitory action of DEX on gastrointestinal transit and its reducing effect on morphine-induced constipation. ANALGESIA: DEX reduced the antinociception induced by mu agonists, morphine, DAMGO and beta endorphin whereas the steroid exerted little or no influence on the antinociception induced by a delta1 agonist, DPDPE and delta2 agonist deltorphin II. DEX potentiated the antinociception induced by the K agonist, U50,488. Cycloheximide, a protein synthesis inhibitor, prevented the antagonism by DEX of responses to the mu opioid agonists. Finally, i.c.v. injection of DEX significantly reduced morphine analgesia in Swiss mice whereas no effects were observed in DBA/2J and C57BL/6 mice. In addition, i.p. injection of DEX significantly reduced morphine analgesia in all three strains. Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the mu. receptor level, while delta and K receptors are modulated in different ways. These results, particularly the effects of drug interaction for i.c.v. administration, strongly confirm a central site for DEX and RU 38486 action as well as the use of different genetic strains may provide a useful approach for studying DEX-morphine analgesia interaction.     

The contribution of comparative studies in inbred strains of mice to the understanding of the hyperactive phenotype

Attention-deficit hyperactivity disorder (ADHD) is a highly prevalent childhood psychiatric disorder characterized by impaired attention, excessive motor activity and impulsivity. Converging evidence, suggests a primary role of disturbances in brain dopamine (DA) transmission and a role of genetic factors in its pathology. Inbred provide a well-defined and stable genotype for analysis. C57BL/6 (C57) and DBA/2 (DBA) mice are amongst the most studied inbred strains in the behavioral pharmacology of DA, and they differ in several parameters of the DA system that relate directly to behavioral differences. These strains also exhibit several qualitatively different behavior patterns that rely on separate DA networks (e.g. mesoaccumbens vs. nigrostriatal) and on different modes of inheritance. C57 mice are good learners in most tasks also involving associative learning but are totally unable to learn active avoidance although being very active. Moreover, C57 mice show greater novelty-induced locomotor activity than DBA, which is modulated strongly by DA neurons in the ventral tegmental area (VTA) region. Pharmacological studies also indicate a facilitated mesoaccumbens DA transmission in C57 mice when compared to DBAs. Increased density of D2 autoreceptors located on VTA neurons, and lower D2 postsynaptic receptors in the NAS were observed in DBA relative to C57. Activation of D2 autoreceptors inhibits impluse flow, synthesis, and release rates of DA neurons. As would be predicted from their higher D2 autoreceptor: DBA compared to C57 mice show reduced DA synthesis and release within the mesoaccumbens DA system when challenged with DA direct agonists. However, DBA mice are by fare more susceptible than C57s to stress-induced enhanced mesoaccumbens DA release and in stressful situation, they show sustained active behavioral responses whilst C57 adopt extremely passive responses (behavioral despair). Finally, chronic or repeated stress promote opposite adaptation of VTA DA autoreceptors in the two strains and render the hypoactive DBAs as active as the C57 mice. These results indicate that a complex interaction between genetic and environmental factors controls, mesoaccumbens DA functioning and hyperactive phenotype.     

Morphine has an antinociceptive effect through activation of the okadaic-acid-sensitive Ser/Thr protein phosphatases PP 2 A and PP5 estimated by tail-pinch test in mice

Although the serine/threonine protein kinases involved in the pharmacological action of morphine are well recognized, the critical contribution of serine/threonine protein phosphatase (PP) has been appreciated on to a slight degree. We examined the involvement of subtypes of serine/threonine protein phosphatase (PP) in the antinociceptive effect of morphine in mice. The antinociceptive effect of subcutaneously administered morphine was attenuated by simultaneously intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection of okadaic acid (OA), a PP inhibitor. To reveal which subtypes of PPs participated in the antinociceptive effect of morphine, mice received i.c.v. or i.t. injections of antisense oligodeoxynucleotide (AS-ODN) directed against either the PP 2 A or PP5 subtypes of PPs before assessment of morphine-induced antinociception. Pretreatment with AS-ODN against PP 2 A or PP5 via each route weakened the antinociceptive effect of morphine, accompanied by reduction of expression levels of PP in the periaqueductal gray (PAG) and the spinal cord. Subcutaneously administered morphine increased activity of OA-sensitive PPs in the PAG and the spinal cord in a dose-dependent manner; this was prevented by concurrent administration of naloxone. These results suggest that PP 2 A and PP5 are involved in the antinociceptive effect of morphine in mice.     

Neuronal protein kinase C gamma-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine

Repeated administration of morphine induced a time-dependent inhibition of the morphine-induced antinociceptive action, indicating the development of tolerance to morphine. We demonstrated that mice tolerant to morphine exhibited a significant increase in the level of protein kinase Cgamma-like immunoreactivity (PKCgamma-IR) in the dorsal horn of the spinal cord. The PKCgamma-IR was exclusively colocalized with the neuron-specific markers neuronal nuclei (NeuN) and microtubule associated protein 2ab (MAP2ab). Here we found a dramatic increase in reactive astrocytes in the dorsal horn of the spinal cord following repeated treatment with morphine, as characterized by the increase and morphological changes in glial fibrillary acidic protein (GFAP)-positive cells. Furthermore, transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the mouse GFAP promoter displayed enhanced levels of EGFP expression after repeated treatment with morphine. Under these conditions, mice lacking the PKCgamma gene failed to show any changes in astroglial hypertrophy or proliferation after repeated treatment with morphine. These findings strongly support the idea that the sustained activation of neuronal PKCgamma is implicated in the increased levels of reactive astrocytes in the dorsal horn of the spinal cord following repeated treatment with morphine. This neuron-glia communication may lead to the development of tolerance to morphine-induced antinociception.     

Cross-tolerance between D-amino acids and morphine in mice

The effects of morphine administration on analgesia and running activity were studied in DBA/2 (DBA) and C57BL/6 (C57) mice respectively, injected withd-amino acids (d-AA) for 5 days. The results indicate a clear cross-tolerance betweend-AA and morphine in DBA mice when analgesia is considered, while the stimulating effect of the opiate is not modified afterd-AA pretreatment in the C57 strain.     

Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors

Previous studies have demonstrated the virtual lack of analgesia in mu opioid receptor knockout mice after systemic administration of morphine. Thus, it has been suggested that analgesic actions of morphine are produced via the mu opioid receptor, despite its ability to bind to kappa and delta receptors in vitro. However, it is not clear whether the results of these studies reflect the effect of morphine in the spinal cord. In the present study, we report study of the analgesic actions of spinally-administered morphine and other opioid receptor agonists in mu opioid receptor knockout and wild type mice. Morphine produced a dose-dependent antinociceptive effect in the tail flick test in the knockout mice, although higher doses were needed to produce antinociception than in wild type mice. The antinociceptive effect of morphine was completely blocked by naloxone (a non-selective opioid antagonist) and nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist), but not by naltrindole (a selective delta-opioid receptor antagonist). U-50,488H (a selective kappa-opioid receptor agonist) also produced a dose-dependent antinociceptive effect in knockout mice but presented lower analgesic potency in knockout mice than in wild type mice. Analgesic effects of [d-Pen2,d-Pen5]enkephalin (DPDPE, a selective delta-opioid receptor agonist) were observed in wild type mice but abolished in knockout mice. SNC80 (a selective delta-opioid receptor agonist) was not antinociceptive even in wild type mice. The present study demonstrated that morphine can produce thermal antinociception via the kappa opioid receptor in the spinal cord in the absence of the mu opioid receptor. Lower potency of U50,488H in mu opioid receptor knockout mice suggests interaction between kappa and mu opioid receptors at the spinal level.     

Normal levels of histamine and Tele-Methylhistamine in mast cell-deficient mouse brain

To determine the contribution by mast cells to the brain content of histamine (HA) and its metabolite tele-methylhistamine (t-MH), the number of mast cells, as well as the levels of HA and t-MH were measured in brain regions of mast cell-deficient (W/W^v) and control (+/+) mice. In agreement with earlier studies, mast cells were identified in control mouse brains, whereas W/W^v) brains were devoid of mast cells. Contrary to earlier studies, no differences between these strains were found in the HA levels of any brain region, implying that mouse brain mast cells do not contribute significantly to brain HA levels. Braint t-MH levels were also not different between strains, except in hypothalamus, where W/W^v levels were higher; a significantly smaller W/W^v hypothalamus accounted for this difference. It is not certain that such differences are due to the absence of mast cells, since the W/W^v mutant is pleiomorphic, and the biochemical nature of this mutation remains uncertain. However, the absence of mast cells and presence of HA in the W/W^v mouse brain is direct evidence for the existence of non-mast cell HA in the brain. These results also show that mouse brain t-MH levels are predictive of non-mast cell HA in brain.     

Spinal mechanisms of delta 9-tetrahydrocannabinol-induced analgesia.

Cannabinoids have been demonstrated to be effective antinociceptive agents when given intravenously. In order to determine whether spinal antinociception can be achieved while minimizing psychotomimetic properties, the pharmacological activity of delta 9-tetrahydrocannabinol (THC) was evaluated after intrathecal injection in male ICR mice. Although delta 9-THC produced potent antinociception, it also caused hypoactivity, hypothermia, and catalepsy. Intrathecal administration of delta 9-THC in mice which had their spinal cord transected at T12 also produced potent antinociception suggesting a spinal component to the antinociceptive effect. Biodispositional studies of [3H]delta 9-THC demonstrated that brain levels of the drug following intrathecal injection in spinally transected animals were not sufficient to produce the antinociceptive effect. These studies suggest the involvement of a spinal component in the antinociceptive action of the cannabinoids.     

Brain histamine turnover enhanced by footshock

When footshock was given to mice at 15-s intervals for 30–120 min, there was a significant increase in the brain level of the tele-methyl-histamine (t-MH), a predominant metabolite of brain histamine (HA). This footshock-induced elevation of the t-MH level also occurred in mice pretreated with pargyline but not in mice pretreated with metoprine. The footshock facilitated the HA depletion induced by α-fluoromethylhistidine. These results suggest that footshock increases the brain HA turnover.     

The relationship between morphine analgesia and the activity of bulbo-spinal serotonergic system as studied by tolerance phenomenon

The effect of various doses of acute morphine on both analgesia and 5-hydroxytryptamine (5-HT) synthesis in the brain and the spinal cord has been studied in rats rendered tolerant by chronic administration of the analgesic. In morphine-tolerant rats, the incorporation of tritiated-L-tryptophan (TRP) in the brain and the spinal cord was higher than in non-tolerant rats, but there was no significant difference in the synthesis rate of the newly formed 5-HT between the two groups. An acute dose of morphine (10 mg/kg) which induced a powerful analgesia and a large increase in 5-HT synthesis in non-tolerant rats, did not produce analgesia nor changes in 5-HT synthesis in tolerant rats. Higher acute doses of morphine which restored analgesia in tolerant rats, induced a discrete increase in [3H]TRP incorporation and a marked increase in 5-HT synthesis in the spinal cord of these animals. The same doses significantly increased [3H]TRP incorporation in the forebrain but did not modify 5-HT synthesis. These results show that tolerance to morphine is associated with a decrease in the effects of the drug on 5-HT synthesis in the spinal cord and the brain and tend further support to the hypothesis that an enhancement of 5-HT synthesis in the spinal cord, induced independently of modifications of the availability of TRP, is associated with the analgesic effect of morphine.     

Region-specific changes in NMDA receptor mRNA induced by chronic morphine treatment are prevented by the co-administration of the competitive NMDA receptor antagonist LY274614

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.     

Strain differences in the development of susceptibility to audiogenic seizures after acoustic priming but not after hearing loss

Either exposure to an initial auditory stimulus (IAS) or external ear plugging (EEP) was used to produce susceptibility to audiogenic seizures in C57BL/6Bg and DBA/1Bg mice. After the IAS, increments in seizure susceptibility occurred by 5 h in C57BL/6Bg mice and by 24 h in DBA/1Bg-ras mice, whereas after EEP, increments in seizure susceptibility occurred by 48 h in C57BL/6Bg and by 24 h in DBA/1Bg-ras mice. Because both the IAS and EEP produce hearing loss, the strain differences in the effect of the IAS on the development of susceptibility and the strain similarities in the effect of the EEP on the development of susceptibility support the hypothesis that acoustic priming in the C57BL/6Bg at 19 days of age involves another mechanism in addition to that of hearing loss and disuse supersensitivity. It was suggested elsewhere that the other mechanism is mediated by a post-IAS decrease in the concentration of brain γ-aminobutyric acid and requires brain protein synthesis for a brief period post-IAS.     

Effects of Aquaporin 4 Deficiency on Morphine Analgesia and Chronic Tolerance: A Study at Spinal Level

Recent reports showed that aquaporin 4 (AQP4) deficiency potentiated morphine analgesia but attenuated chronic morphine-induced tolerance in hot-plate test, predominantly reflecting supraspinal pain response. The present study investigated the effects of AQP4 deficiency on morphine analgesia and tolerance using tail flick test, primarily reflecting spinal response. It was found that (1) chronic morphine treatment resulted in decreased expression of spinal AQP4 in mice detected by Western blotting analysis; (2) in tail flick test, AQP4 knockout mice displayed significant impaired morphine analgesia without influencing the progress of chronic tolerance; and (3) the expressions of mu-opioid receptor (MuOR) and glutamate transporter 1 (GLT-1) in AQP4 knockout mice spinal cord were lower than those in wild-type mice, whereas chronic morphine-induced alteration characteristics of spinal MuOR or GLT-1 expression were not affected by AQP4 deficiency. In conclusion, AQP4 deficiency attenuated morphine acute antinociception but did not affect chronic tolerance in tail flick test, implying a role for spinal AQP4 in morphine analgesia but not in chronic tolerance.     

Social conflict-induced changes in nociception and β-endorphin-like immunoreactivity in pituitary and discrete brain areas of C57BL/6 and DBA/2 mice

The present study characterizes the time course of social conflict analgesia and its reversibility by opioid antagonist drugs in the C57BL/6 and DBA/2 inbred strains of mice and examines the relationship between alterations in brain and pituitary levels of β-endorphin-like immunoreactivity (β-ELIR) and the antinociception elicited by social stress. Data revealed statistically strain differences in regard to β-ELIR in control animals. The pituitary content of β-ELIR was higher in DBA/2, while the values in the periaqueductal grey (PAG) and in the amygdala were higher in C57BL/6 mice. No interstrain differences were found in the hypothalamus. Exposure to 50 attack bites resulted in a 6-fold higher analgesia in DBA/2 mice and in a strain-dependent fall of ELIR in pituitary (27%) and PAG (23%). PAG but not pituitary β-ELIR levels in C57BL/6 mice correlated positively with the increase in tail-flick latency after attack. Mere confrontation with a non-aggressive opponent failed to induce analgesia and was associated in C57BL/6 mice with a significant reduction in the β-ELIR content of both the pituitary and the PAG. The data are discussed in terms of genotype-dependent sensitivity of the β-endorphin system to stress and its relation to analgesia.