Withdrawal from dependence upon butorphanol uniquely increases kappa(1)-opioid receptor binding in the rat brain

Changes in kappa(1)-opioid receptor binding have been implicated in the development of dependence upon and withdrawal from butorphanol. Autoradiographic characterization of binding for brain kappa(1)-([3H]CI-977), mu-([3H]DAMGO), and delta-([3H]DPDPE) opioid receptors was performed in rats undergoing naloxone-precipitated withdrawal from dependence upon butorphanol or morphine. Dependence was induced by a 72h i.c.v. infusion with either butorphanol or morphine (26nmol/microl/h). Withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 microl/rat), administered 2h following cessation of butorphanol or morphine infusion. During withdrawal from butorphanol, but not morphine, kappa(1)-opioid receptor binding was increased significantly in the frontal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus, posterior paraventricular thalamic nucleus, ventral tegmental area and locus coeruleus. In contrast, mu-opioid receptor binding decreased in these brain regions in naloxone-precipitated withdrawal from morphine, but not butorphanol, while binding for delta-opioid receptors was altered in both withdrawal groups. The brain kappa(1)-opioid receptor appears to be more directly involved in the development of physical dependence upon, and the expression of withdrawal from, butorphanol, as opposed to the prototypical opioid analgesic, morphine.     

Enhanced binding of nor-binaltorphimine to kappa-opioid receptors in rats dependent on butorphanol

Autoradiographic characterization of binding for brain kappa(1) ([(3)H]CI-977) and kappa(2) ([(3)H]bremazocine) in the presence of DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin), DPDPE ([D-Pen(2), D-Pen(5)]-enkephalin), and U-69,593 opioid receptors, in the presence of different concentrations of a selective unlabeled kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI), was performed in rats in which dependence on or withdrawal from butorphanol had been established. Dependence was induced by a 72 hr intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/microl/hr; butorphanol dependent). Butorphanol withdrawal was produced by terminating the infusion of butorphanol in dependent animals. Responses were studied 7 hr following termination (butorphanol withdrawal). IC(50) values from competition studies were estimated by fitting inhibition curves for both kappa(1)- and kappa(2)-opioid receptor assays. In both dependent and withdrawal groups, the IC(50) values obtained against [(3)H]CI-977 or [(3)H]bremazocine with nor-BNI were decreased (ratios of approximately 0.03-0.21 and approximately 0.05-0.42 vs. control, respectively) in brain regions, including frontal cortex, nucleus accumbens, claustrum, dorsal endopiriform nucleus, caudate putamen, parietal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus, posterior paraventricular thalamic nucleus, periaqueductal gray, substantia nigra, superficial gray layer of the superior colliculus, ventral tegmental area, and locus coeruleus, compared with control. These results indicate that, in butorphanol-dependent and butorphanol-withdrawal rats, the brain kappa(1)- and kappa(2)-opioid receptors developed a supersensitivity to antagonist binding.     

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.     

Multiple opioid receptors mediate feeding elicited by mu and delta opioid receptor subtype agonists in the nucleus accumbens shell in rats

The nucleus accumbens, and particularly its shell region, is a critical site at which feeding responses can be elicited following direct administration of opiate drugs as well as micro-selective and delta-selective, but not kappa-selective opioid receptor subtype agonists. In contrast to observations of selective and receptor-specific opioid antagonist effects upon corresponding agonist-induced actions in analgesic studies, ventricular administration of opioid receptor subtype antagonists blocks feeding induced by multiple opioid receptor subtype agonists. The present study examined whether feeding responses elicited by either putative mu ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO)), delta(1) ([D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)) or delta(2) ([D-Ala(2), Glu(4)]-deltorphin (Deltorphin)) opioid receptor subtype agonists administered into the nucleus accumbens shell were altered by accumbens pretreatment with either selective mu (beta-funaltrexamine), mu(1) (naloxonazine), delta(1) ([D-Ala(2), Leu(5), Cys(6)]-enkephalin (DALCE)), delta(2) (naltrindole isothiocyanate) or kappa(1) (nor-binaltorphamine) opioid receptor subtype antagonists. Similar magnitudes and durations of feeding responses were elicited by bilateral accumbens administration of either DAMGO (2.5 microg), DPDPE (5 microg) or Deltorphin (5 microg). DAMGO-induced feeding in the nucleus accumbens shell was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. DPDPE-induced feeding in the accumbens was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. Deltorphin-induced feeding in the accumbens was largely unaffected by accumbens delta(2) antagonist pretreatment, and was significantly enhanced by accumbens mu or kappa(1) antagonist pretreatment. These data indicate different opioid pharmacological profiles for feeding induced by putative mu, delta(1) and delta(2) opioid agonists in the nucleus accumbens shell, as well as the participation of multiple opioid receptor subtypes in the elicitation and maintenance of feeding by these agonists in the nucleus accumbens shell.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

Effects of neonatal cocaine treatment and gender on opioid agonist-stimulated [S]GTPγS binding in the striatum and nucleus accumbens

Prenatal cocaine exposure increases μ-opioid receptor binding in dopaminergic terminal areas and enhances behavioral responsiveness to μ-opioid agonists. We investigated the influence of early postnatal cocaine treatment on in vitro μ- and δ-opioid receptor activation in male and female weanling rats. Pups received subcutaneous injections of either 20 mg/kg cocaine HCl or saline once daily on postnatal days 1 through 5. On postnatal day 25, animals were decapitated and their brains were removed and frozen for later sectioning. Opioid receptor activation was assessed in the striatum and the shell of the nucleus accumbens by autoradiographic analysis of agonist-stimulated [³⁵S]GTPγS binding. Brain sections were incubated in the presence of [³⁵S]GTPγS, GDP, and either the μ-opioid agonist [ -Ala²-N-MePhe⁴-Gly⁵-ol]enkephalin (DAMGO) or the δ-opioid agonist -Pen²-D-Pen⁵-enkephalin (DPDPE). Baseline binding was assessed in the absence of agonist, and nonspecific binding was determined by the addition of unlabeled GTPγS. Film images were quantified using brain mash-calibrated [¹⁴C] standards. Neonatal cocaine treatment had no effect on either baseline or agonist-stimulated [³⁵S]GTPγS binding. However, males exhibited significantly greater activation than females of δ-opioid receptors in both striatum and accumbens shell, regardless of neonatal treatment. These findings indicate a gender difference in δ-opioid receptor function that could mediate behavioral differences in response to opioid agonists.     

Antinociception produced by receptor selective opioids. Modulation of supraspinal antinociceptive effects by spinal opioids

This study evaluated the antinociceptive effects produced when different combinations of supraspinal μ- and δ-opioid agonist were co-administered with spinal μ-, δ-, and κ-opioid agonist. Using the Randall-Selitto paw-withdrawal test, in the rat, changes in nociceptive thresholds were measured following co-administration of sequentially increasing i.c.b. doses of either DAMGO or DPDPE with a low-antinociceptive dose of intrathecal DAMGO, DPDPE, or U50,488H. Antinociceptive synergy (i.e. a more than additive antinociceptive effect) was demonstrated with all of the combinations tested except for supraspinal DPDPE co-administered with spinal DAMGO. The results of this study provide support for the suggestion that supraspinal and spinal antinociceptive mechanisms share, in part, common neural circuits. Marked differences in the overall magnitude of the antinociceptive effects produced by the various combinations of opioid agonists were demonstrated through a secondary analysis of the data. When sequentially increasing i.c.v. doses of DAMGO were administered, significantly larger increases in nociceptive thresholds were observed with co-administration of intrathecal injections of low antinociceptive doses of either DAMGO or U50,488H compared to DPDPE. In contrast, when DPDPE was administered supraspinally, the largest increases in nociceptive thresholds were demonstrated with co-administration of DPDPE at the spinal site. The results of the secondary analysis provide support for the hypothesis that descending antinociceptive control systems activated by supraspinal administration of selective μ- and δ-opioid agonists interact, differently, with spinal μ-, δ, and κ-opioidergic mechanisms.     

Quantitative autoradiographic mapping of μ-, δ- and κ-opioid receptors in knockout mice lacking the μ-opioid receptor gene

Mice lacking the μ-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any μ-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. μ-, δ-, κ₁- and total κ-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [³H]DAMGO (

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-Ala²-MePhe⁴-Gly-ol⁵ enkephalin), [³H]DELT I (

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-Ala² deltorphin I), [³H]CI-977 and [³H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, μ-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from μ-knockout mice there were no detectable μ-receptors in any brain regions and no evidence for μ-receptors derived from another gene. δ-, κ₁- and total κ-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in κ- or δ-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in δ- and κ-receptors throughout the brains of animals deficient in μ-receptors.     

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of spinal opioid analgesia by supraspinal administration of selective opioid antagonists

The effect of intracerebroventricular administration of a selective mu- (CTOP) or delta- (ICI 174,864) opioid receptor antagonist on the antinociceptive effects produced by intrathecal administration of selective mu- (DAMGO), delta- (DPDPE) and kappa- (U50-488H) opioid receptor agonists was evaluated using the Randall-Selitto paw-withdrawal test, in the rat. While the intracerebroventricular administration of CTOP or ICI 174,864, alone, had no effect on nociceptive thresholds, intracerebroventricular administration of CTOP and ICI 174,864 produced marked antagonism of the antinociceptive effects of intrathecal DAMGO. The antinociceptive effects of intrathecal administration of DPDPE or U50,488H were not antagonized by intracerebroventricular administration of CTOP or ICI 174,864. These data suggest that, in the rat, along with the established descending antinociceptive pathways, there is an ascending antinociceptive control mechanism projecting from the spinal cord to the brainstem. The ascending antinociceptive control involves mu- and delta-opioid agonism at supraspinal sites and appears to be mediated selectively by mu-, but not by delta- or kappa-opioid agonism at the spinal level.     

Up-regulation of spinal μ-opioid receptor function to activate G-protein by chronic naloxone treatment

The effects of repeated s.c. administrations of an μ-opioid receptor antagonist naloxone on the G-protein activation induced by μ-opioid receptor agonists [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding. All μ-opioid receptor agonists concentration-dependently increased the [³⁵S]GTPγS binding. The increases of [³⁵S]GTPγS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [³H]DAMGO binding sites with an increase in K_d values in spinal cord membranes, indicating an increase in μ-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an μ-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by μ-opioid receptor agonists with an increase in μ-opioid receptors in membranes of the mouse spinal cord.     

Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat

This study compared the antinociceptive and motor effects produced by intracerebroventricular administration of selective mu- (DAMGO) and delta- (DPDPE) opioid receptor agonists in the rat. Changes in nociceptive thresholds were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Both DAMGO and DPDPE produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. However, in the motor coordination studies, neither opioid agonist produced statistically significant changes in rotarod performance scores. The dissociation of antinociceptive and motor effects at this supraspinal site differs from the strong association between antinociceptive and motor effects produced by intrathecal administration of the same opioid agonists.     

Antinociception produced by receptor selective opioids: modulation of spinal antinociceptive effects by supraspinal opioids

The effect of intracerebroventricular administration of low-antinociceptive doses of selective μ-(DAMGO) or δ-(DPDPE) opioid agonists on the dose-dependent antinociceptive effects produced by intrathecal administration of sequentially increasing doses of selective μ-, δ-, or κ- (U50, 488H) opioid agonists was evaluated, in the rat, using the Randall-Selitto paw-withdrawal test. When DPDPE or U50,488H was administered intrathecally, the low doses of both intracerebroventricular DAMGO and intracerebroventricular DPDPE markedly enhanced the antinociceptive effects of both intrathecal opiods. In contrast, when DAMGO was administered intrathecally, both intracerebroventricular DAMGO and intracerebroventricular DPDPE, administered in low doses, markedly antagonized the antinociceptive effects of the intrathecal opioid. In addition, the intracerebroventricular administration of low-antinociceptive dose of a second μ-opioid agonist, morphiceptin, antagonized the antinociceptive effects of intrathecal morphiceptin. The antagonism of the antinociceptive effects observed with spinal administration of DAMGO is dose-dependent, with the effect observed only at low doses. Furthermore, the antagonism cannot be explained by a reduction in motor deficits produced by intrathecal administration of DAMGO, because there were no differences in motor deficits, measured with an accelerating Rotarod treadmill, between intrathecal DAMGO administered as a single agent or as part of a combination regimen. The differences in antinociceptive effects obtained with the various supraspinal and spinal combinations are discussed in terms of the interactions that may occur between brainstem and spinal opioid receptor sites.     

Quantitative autoradiographic mapping of opioid receptors in the brain of delta-opioid receptor gene knockout mice

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor.     

Neuroanatomical localization ofκ1and κ2 opioid receptors in rat and guinea pig brain

The neuroanatomical localization of kappa opioid receptors in rat and guinea pig brain was determined by quantitative in vitro receptor autoradiography. Our study shows striking differences in kappa 1 and kappa 2 receptor distributions both between species and within each species. In the rat brain, kappa 1 sites (labeled by [3H]U-69,593) are of low density and confined to a small number of structures. These include the claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, midline nuclear group of the thalamus, superficial grey layer of the superior colliculus, and central grey. kappa 2 sites (labeled by [3H]ethylketocyclazocine or [3H]bremazocine under conditions in which mu, delta, and kappa 1 binding was suppressed) are more widely distributed throughout all levels of rat brain. kappa 2 sites occur at high density in the caudate putamen, nucleus accumbens, amygdala, thalamus, and interpeduncular nuclei. In guinea pig brain, kappa 1 sites predominate and are of high density in layers I and VI of the neocortex, claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, and molecular layer of the cerebellum. As in rat brain, kappa 2 sites in guinea pig are more uniformly and widely distributed throughout the brain than are kappa 1 sites. The highest density of kappa 2 sites is in the dorsal parabrachial nucleus, interpeduncular nuclei, mammillary nuclei, and posterior thalamic nuclei. Results from this study demonstrate important interspecies differences in the distribution of kappa 1 and kappa 2 opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antisense oligodeoxynucleotides to the κ-1 receptor block the antinociceptive effects of Δ-THC in the spinal cord

Intrathecal pretreatment of mice with an antisence oligodeoxynucleotide directed against the κ-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as Δ⁹-THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 orΔ⁹-THC. Furthermore, κ-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using κ-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.     

Pharmacological and anatomical evidence of selective mu, delta, and kappa opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [3H]DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), [3H]DPDPE (2-D-penicillamine-5-D-penicillamine-enkephalin), [3H]DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and [3H](-)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opioid receptors in rat brain. The results from saturation, competition, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [3H]DAGO and [3H]DPDPE selectively labelled mu and delta binding sites, respectively. In contrast, [3H]DSLET was found to be relatively non-selective, and labelled both mu and delta sites. [3H]Bremazocine was similarly non-selective in the absence of mu and delta ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the mu and delta sites, [3H]bremazocine did label kappa sites selectively. The high affinity [3H]bremazocine binding sites showed a unique distribution with relatively dense kappa labelling in the hypothalamus and median eminence, areas with extremely low mu and delta binding. These results point to the selectivity, under appropriate conditions, of [3H]DAGO, [3H]DPDPE and [3H]bremazocine and provide evidence for the differential distribution of mu, delta, and kappa opioid receptors in rat brain.     

Anti-nociceptive effect of neuropeptide Y in the nucleus accumbens of rats: an involvement of opioid receptors in the effect

The present study investigated the effect of neuropeptide Y on nociception in the nucleus accumbens of rats. Intra-nucleus accumbens administration of neuropeptide Y induced dose-dependent increases in the hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation in rats. There were no significant changes in the HWL to both stimulation during 60 min after the administration of NPY to outside of the nucleus accumbens. The anti-nociceptive effect of NPY was blocked by subsequent intra-nucleus accumbens injection of the Y1 receptor antagonist neuropeptide Y 28–36, indicating that Y1 receptor is involved in the neuropeptide Y-induced anti-nociception in the nucleus accumbens. Furthermore, the anti-nociceptive effect of neuropeptide Y was attenuated by intra-nucleus accumbens administration of the opioid antagonist naloxone, suggesting an involvement of the endogenous opioid system in the neuropeptide Y-induced anti-nociception in the nucleus accumbens of rats. Moreover, the neuropeptide Y-induced anti-nociception was attenuated by following intra-nucleus accumbens injection of the selective opioid antagonists nor-binaltorphimine and β-funaltrexamine, but not by naltrindole, illustrating that μ- and κ-opioid receptors, not the δ-opioid receptor, were involved in the neuropeptide Y-induced anti-nociception in the nucleus accumbens of rats.     

κ-opioid agonist stimulated regional distribution of [S]GTPγs binding in butorphanol continuously infused rat

Butorphanol is a mixed agonist/antagonist opioid analgesic agent, which exerts its effects mainly by interaction with the kappa-opioid receptor. Opioid receptors are coupled to G proteins of G(i)/G(o) family, and recently a decrease in micro-opioid activation of G proteins has been reported in specific brainstem nuclei after chronic morphine administration. The influence of centrally administered butorphanol on agonist-stimulated G protein coupling was examined in the rat brain, using in situ guanylyl-5'-O-(gamma-[(35)S]thio)-triphosphate (GTPgammaS) binding autoradiography. Rats were treated with butorphanol (26 nmol/microl/h) by intracerebroventricular infusion via osmotic minipumps for 3 days. The distribution of [(35)S]GTPgammaS binding in the brain 7 h after the termination of butorphanol infusion was measured in the presence or absence of the selective kappa-opioid agonist, U-50,488. This agonist significantly increased [(35)S]GTPgammaS binding in the parietal cortex, caudate putamen, thalamus, and central gray of control rats, but not in those regions of the butorphanol-infused animals. These results suggest that chronic administration of butorphanol developed tolerance and abolished U-50,488 activation of G proteins in these brain areas.     

Neuroanatomical and neuropharmacological study of opioid pathways in the mesencephalic tectum: effect of mu(1)- and kappa-opioid receptor blockade on escape behavior induced by electrical stimulation of the inferior colliculus

Deep layers of the superior colliculus (DLSC), the dorsal and ventral periaqueductal gray matter (PAG), and inferior colliculus (IC) are midbrain structures involved in the generation of defensive behavior. beta-Endorphin and Leu-enkephalin are some neurotransmitters that may modulate such behavior in mammals. Light microscopy immunocytochemistry with streptavidin method was used for the localization of the putative cells of defensive behavior with antibodies for endogenous opioids in rat brainstem. Midbrain structures showed positive neurons to beta-endorphin and Leu-enkephalin in similar distributions in the experimental animals, but we also noted the presence of varicose fibers positive to endogenous opioids in the PAG. Neuroanatomical techniques showed varicose fibers from the central nucleus of the inferior colliculus to ventral aspects of the PAG, at more caudal levels. Naloxonazine and nor-binaltorphimine, competitive antagonists that block mu(1)- and kappa-opioid receptors, were then used in the present work to investigate the involvement of opioid peptide neural system in the control of the fear-induced reactions evoked by electrical stimulation of the neural substrates of the inferior colliculus. The fear-like responses were measured by electrical stimulation of the central nucleus of the inferior colliculus, eliciting the escape behavior, which is characterized by vigorous running and jumping. Central administration of opioid antagonists (2.5 microg/0.2 microl and 5.0 microg/0.2 microl) was performed in non-anesthetized animals (Rattus norvegicus), and the behavioral manifestations of fear were registered after 10 min, 2 h, and 24 h of the pretreatment. Naloxonazine caused an increase of the defensive threshold, as compared to control, suggesting an antiaversive effect of the antagonism on mu(1)-opioid receptor. This finding was corroborated with central administration of nor-binaltorphimine, which also induced a decrease of the fear-like responses evoked by electrical stimulation of the inferior colliculus, since the threshold of the escape behavior was increased 2 and 24 h after the blockade of kappa-opioid receptor. These results indicate that endogenous opioids may be involved in the modulation of fear in the central nucleus of the inferior colliculus. Although the acute treatment (after 10 min) of both naloxonazine and nor-binaltorphimine causes nonspecific effect on opioid receptors, we must consider the involvement of mu(1)- and kappa-opioid receptors in the antiaversive influence of the opioidergic interneurons in the dorsal mesencephalon, at caudal level, after chronic (2-24 h) treatment of these opioid antagonists. The neuroanatomical study of the connections between the central nucleus of the inferior colliculus and the periaqueductal gray matter showed neuronal fibers with varicosities and with terminal bottons, both in the pericentral nucleus of the inferior colliculus and in ventral and dorsal parts of caudal aspects of the periaqueductal gray matter.