Μ-opioid Receptor Knockout Mice Research Articles

Effects of morphine on pentobarbital-induced responses in μ-opioid receptor knockout mice

Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using μ-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in μ-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the μ-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in μ-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through μ-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in μ-opioid receptor knockout mice than in wild-type mice.

Region specific increase of dopamine receptor D1/D2 mRNA expression in the brain of μ-opioid receptor knockout mice

Previous pharmacological studies have indicated the possible existence of functional interactions between opioidergic and dopaminergic neurons in the CNS. In this study, the expression of mRNAs encoding dopamine receptor D1/D2 was examined to investigate whether there is a change in the dopamine pathway of mice lacking the μ-opioid receptor by in situ hybridization technique. In the μ-opioid receptor knockout mice, the expression of dopamine receptor D1 mRNA was increased in the olfactory tubercle, nucleus accumbens, caudate putamen, and the layer VI of the neocortex compared with that of wild-type mice. The expression of dopamine receptor D2 mRNA was also increased in the olfactory tubercle, caudate putamen, and the nucleus accumbens of μ-opioid receptor knockout mice. These results indicate that there are compensational changes in the dopaminergic systems of μ-opioid receptor knockout mice.

Room 302, 10/17/2000 9: 00 AM - 10: 30 AM (PD) Anesthetic Potency in μ-Opioid Receptor Knockout Mice

<b>Room 302, 10/17/2000 9: 00 AM - 10: 30 AM (PD) Anesthetic Potency in μ-Opioid Receptor Knockout Mice</b>

Comparison of G-protein activation in the brain by μ-, δ-, and κ-opioid receptor agonists in μ-opioid receptor knockout mice

Mice lacking the μ-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for μ-, δ-, and κ-opioid receptors in μ-opioid receptor knockout mice. Selective agonists of μ- (DAMGO), δ- (DPDPE), and κ- (U-69,593) opioid receptors stimulated [ 35S]GTPγS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [ 35S]GTPγS binding in these regions of μ-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a μ-opioid receptor agonist in μ-opioid receptor knockout mice, and demonstrated that coupling of the κ-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the δ-opioid receptor agonist, DPDPE, was reduced in the μ-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.

Stress-induced analgesia in μ-opioid receptor knockout mice reveals normal function of the δ-opioid receptor system

Stress-induced analgesia (SIA) was examined in wildtype and μ-opioid receptor knockout mice. We used thermal paw withdrawal (TPW) latency following a continuous 3-min swim in 20°C water, and found a significant increase in TPW latency in both wild-type and knockout mice. Pre-treatment prior to the swim with naltrindole, a selective δ-opioid receptor antagonist, blocked the increase in TPW latency in knockout mice. These results demonstrate an intact δ-receptor-mediated function of a physiologically-released endogenous agonist in the μ-opioid receptor knockout mouse. The present findings are in contrast with previous reports that analgesia induced by exogenous delta agonists is reduced in the knockout mice.

Involvement of μ-opioid receptors in potentiation of apomorphine-induced climbing behavior by morphine: studies using μ-opioid receptor gene knockout mice

The present study examined the hypothesis that μ-opioid receptors contribute to a behavioral stimulation produced by stimulation of dopamine receptors by comparing responses in μ-opioid receptor knockout and wild type mice. Apomorphine-induced climbing behavior was augmented by 65%, in wild type mice, but not in μ-knockout, following subcutaneous administration of morphine (15 mg/kg). Moreover, pretreatment with either naloxone (an opioid receptor antagonist) or haloperidol (a mixed D 1/D 2 receptor antagonist) eliminated the enhancement by morphine of climbing behavior in wild type mice. These results indicate that expression of μ-opioid receptors plays an important role in the enhancement of climbing behavior induced by the dopamine receptor agonist, apomorphine. Furthermore, this augmentation is mediated by interaction between dopamine and μ-opioid receptors.

δ-Opioid receptor agonists produce antinociception and [ 35S]GTPγS binding in μ receptor knockout mice

We examined the effects of [ d-Pen 2, d-Pen 5]enkephalin (DPDPE), [ d-Ala 2,Glu 4]deltorphin (DELT), and (+)-4-[(α R)-α((2 S,5 R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N, N-diethylbenzamide (SNC80) on [ 35S]GTPγS binding in brain membranes prepared from μ-opioid receptor knockout (−/−) mice. The potency and maximal response ( E max) of these agonists were unchanged compared to control mice. In contrast, while the potency of [ d-Pen 2,pCl-Phe 4, d-Pen 5]enkephalin (pCl-DPDPE) was not significantly different, the E max was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in −/− mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in −/− compared to control mice, respectively. Reduced DPDPE antinociceptive potency in −/− mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from −/− mice, demonstrate that these agonists require μ-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between −/− and wild type mice, we conclude that the μ-opioid receptor is not a critical component of δ-opioid receptor function.

Enhanced δ-opioid receptor-mediated antinociception in μ-opioid receptor-deficient mice

Inflammatory hyperalgesia was induced in wild-type, heterozygous and μ-opioid receptor knockout mice after an intraplantar injection of complete Freund's adjuvant. μ-Opioid receptor knockout mice exhibited faster recovery from hyperalgesia as compared to heterozygous ( P<0.05) and wild-type ( P<0.01) mice. Naloxone restored hyperalgesia in all genotypes. Naltrindole (δ-opioid receptor-selective antagonist) partially restored the hyperalgesia only in μ-opioid receptor knockout mice ( P<0.001). Nor-binaltorphimine (κ-opioid receptor-selective antagonist) had no effect. The μ-opioid receptor-selective agonist, [ d-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAMGO), reduced the hyperalgesia in heterozygous and wild-type but not in μ-opioid receptor knockout mice while U69,593 {(+)-(5α,7α,8β)- N-methyl- N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide, κ-opioid receptor-selective} produced similar effects in all mice. The δ-opioid receptor-selective agonists, [ d-Pen 2, d-Pen 5]enkephalin (DPDPE) and deltorphin ([ d-Ala 2]deltrophin-II), produced significantly greater antihyperalgesia in knockout mice ( P<0.05). The findings suggest that μ-opioid receptors may be involved in the persistence of inflammatory hyperalgesia and that a δ-opioid receptor-mediated compensatory mechanism in the absence of the μ-opioid receptor is activated by persistent hyperalgesia.

The μ-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in μ-opioid receptor knockout mice

There appear to be different relationships between μ-opioid receptor densities and the acute and neuroadaptive μ-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating μ-opioid receptor knockout mice allows studies of μ-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the μ-opioid receptor gene. We now report that the binding of μ-opioid receptor ligand, [ 3H][D-Ala 2,NHPhe 4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous μ-opioid receptor knockout mice and eliminated in homozygous μ-opioid receptor knockout mice. The endogenous μ-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [ 35S]guanosine-5′- o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous μ-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The μ-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of μ-opioid agonists. These data support the notion that there are limited physiological μ-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.

Visceral chemical nociception in mice lacking μ-opioid receptors: effects of morphine, SNC80 and U-50,488

Writhing responses to intraperitoneal acetic acid administration and their modulation by μ-, κ- and δ-opioid receptor agonists were compared in wild-type and μ-opioid receptor knockout mice. Unpretreated homozygous knockout mice displayed less writhing than wild-type mice. U-50,488 [ trans-3,4-dichloro- N-methyl- N-[2-(1-pyrolidinyl)cyclohexyl]-benzeneacetamide]) reduced writhing responses in wild-type and knockouts. Morphine and SNC80 [(+)-4-[9-α- R)-α-(2 S,5 RO-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N, N-diethylbenzamide] were effective in wild-type mice but ineffective in knockouts. μ-opioid receptors appear to play important roles in responses to this visceral nociceptive stimulus and its modulation by μ- and δ-opioid receptor agonists.

MU-opioid receptor-knockout mice: role of μ-opioid receptor in morphine mediated immune functions

The role of the μ-opioid receptor in immune function was investigated using μ-opioid receptor knockout mice (MOR-KO). Morphine modulation of several immune functions, including macrophage phagocytosis and macrophage secretion of TNF-α, was not observed in the MOR-KO animals, suggesting that these functions are mediated by the classical μ-opioid receptor. In contrast, morphine reduction of splenic and thymic cell number and mitogen-induced proliferation were unaffected in MOR-KO mice, as was morphine inhibition of IL-1 and IL-6 secretion by macrophages. These latter results are consistent with morphine action on a naloxone insensitive morphine receptor, a conclusion supported by previous studies characterizing a nonopioid morphine binding site on immune cells. Alternatively, morphine may act either directly or indirectly on these cells, by a mechanism mediated by either delta or kappa opioid receptors.

No heroin or morphine 6β-glucuronide analgesia in μ-opioid receptor knockout mice

Recent reports suggest that heroin and its metabolite morphine 6β-glucuronide can produce analgesia independent of the morphine-preferring μ-opioid receptor. We have tested heroin and morphine 6β-glucuronide analgesia in wild-type, homozygous and heterozygous μ-opioid receptor knockout mice. Homozygotes display no heroin or morphine 6β-glucuronide analgesia. Heterozygous mice with one μ-opioid receptor gene copy reveal reduced heroin and morphine 6β-glucuronide analgesia. The μ-opioid receptor-dependence of heroin and morphine 6β-glucuronide fails to support a requirement for a heroin-specific opiate receptor subtype.

μ-Opioid receptor-knockout mice: the role of μ-opioid receptor in gastrointestinal transit

The role of μ-opioid receptor in gastrointestinal transit was investigated using μ-opioid receptor knockout mice (MOR-KO). Our result establishes unequivocally that inhibition of GI transit by morphine is a μ-opioid receptor mediated function. In addition, we show that neither δ nor κ receptor agonist given supraspinally or peripherally are able to inhibit GI transit in MOR-KO animals. It was interesting to observe that basal GI motility was lower in MOR-KO (−/−) compared to heterozygous (±) and wild type (+/+) animals.

The μ-opioid receptor is necessary for [ d-Pen 2, d-Pen 5]enkephalin-induced analgesia

Interactions between δ-opioid receptors and morphine-preferring μ-opioid receptor subtypes have been suggested. Availability of transgenic μ-opioid receptor knockout mice allows assessment of μ-opioid receptor roles in the analgesia produced by the classical δ-opioid receptor agonist [ d-Pen 2, d-Pen 5]enkephalin (DPDPE) in hot-plate and tail-flick tests. DPDPE analgesia was dramatically reduced in μ-opioid receptor knockout mice in a gene-dose-dependent fashion. The analgesia induced by this classic δ-opioid receptor agonist depends on intact μ-opioid receptors, suggesting that selective δ-opioid receptor drugs may require μ-opioid receptor occupancies for full efficacy.