Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

γ-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats

Food intake is significantly increased by administration of μ-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (μ, δ₁, δ₂ or κ₁) or dopaminergic (D₁) receptor antagonists in the nucleus accumbens shell reduce μ opioid agonist-induced feeding. Selective GABA_A (muscimol) and GABA_B (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA_A (bicuculline) and GABA_B (saclofen) antagonists. The present study investigated whether feeding elicited by the μ-selective opioid agonist, [ -Ala²,NMe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA_A or GABA_B antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA_A or GABA_B agonists in the nucleus accumbens shell. Feeding elicited by the μ-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA_A (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or μ-selective opioid antagonists. In contrast, μ opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA_B (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA_A and GABA_B receptor subtype antagonists differentially affect feeding elicited by μ opioid receptor agonists within the nucleus accumbens shell in rats.     

Different central opioid receptor subtype antagonists modify maltose dextrin and deprivation-induced water intake in sham feeding and sham drinking rats

Different central opioid receptor subtypes participate in the mediation of intakes of simple (sucrose: μ, κ₁ and complex (maltose dextrin: μ) carbohydrates as well as deprivation-induced water intake (μ) under real-feeding and real-drinking conditions. An identical pattern of μ and κ₁ mediation of sucrose intake was observed in sham-feeding rats as well, suggesting their actions on orosensory mechanisms supporting sucose intake. The present study examined whether centrally administered general (naltrexone: 1–50 μg), μ (β-funaltrexamine: 1–20 μg), μ₁ (naloxonazine: 50 μg), κ₁ (nor-binaltorphamine: 1–20 μg), δ₁ ([d-Ala², Leu⁵, Cys⁶]-enkephalin: 10–40 μg) or δ₂2 (naltrindole isothiocyanate: 20 μg) opioid subtype antagonists altered either maltose dextrin (10%) intake during sham feeding or deprivation (24 h)-induced water intake during sham drinking in rats with gastric fistulas. Sham feeding significantly increased maltose dextrin intake (180%) and sham drinking significantly increased deprivation-induced water intake (256%) over a 60 min time course. Naltrexone significantly and dose-dependently reduced maltose dextrin intake (78%) in sham feeding rats, and deprivation-induced water intake (51%) in sham drinking rats. Maltose dextrin intake in sham feeding rats was significantly reduced by either κ₁ (69%) or δ₁ (59%) opioid antagonism, was significantly increased by μ₁ antagonism (43%), and was not significantly affected by either μ or δ₂ opioid antagonism. Deprivation-induced water intake in sham drinking rats was significantly reduced by either μ (41%), μ₁ (28%), δ₁ (48%) or δ₈ (28%) opioid antagonism, but was not significantly affected by κ₁ opioid antagonism. The difference in opioid receptor subtype mediation of maltose dextrin intake in real feeding and sham feeding conditions suggest that κ₁ and δ₁ receptors are involved in the orosensory mechanisms supporting maltose dextrin intake, while μ receptors are involved in the ingestive and post-ingestive mechanisms supporting maltose dextrin intake. The different patterns of opioid involvement in sucrose and maltose dextrin intake in sham feeding and real feeding conditions provide further support for the hypothesis that at least two different carbohydrate taste systems exist. The difference in opioid receptor subtype mediation of deprivation-induced water intake in real drinking and sham drinking conditions may reflect the removal in the sham drinking condition of a μ-mediated prerestorative satiety mechanism, and the unmasking of other opioid-mediated signalling mechanisms.     

Intrathecal dynorphin A1–13 and dynorphin A3–13 reduce rat spinal cord blood flow by non-opioid mechanisms

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A_1–13) and Dyn A_3–13 on rat brain and spinal cord blood flows and cardiac output. Dyn A_1–13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A_3–13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.     

Differential modulation of angiotensin II and hypertonic saline-induced drinking by opioid receptor subtype antagonists in rats

Opioid modulation of ingestion includes general opioid antagonism of different forms of water intake, μ₂ receptor modulation of deprivation-induced water intake and δ₂ receptor modulation of saccharin intake. Water intake is stimulated by both central administration of angiotensin II (ANG II) and peripheral administration of a hypertonic saline solution; both responses are reduced by general opioid antagonists. The present study examined whether specific opioid receptor subtype antagonists would selectively alter each form of water intake in rats. Whereas systemic naltrexone (0.1–2.5 mg/kg, s.c.) reduced water intake induced by either peripheral ANGII (500 μg/kg, s.c.) or hypeptonic saline (3 ml/kg, 10%), intracerebroventricular (i.c.v.) naltrexone (1–50 μg) only inhibited central ANGII (20 ng)-induced hyperdipsia. Both forms of drinking were significantly and dose-dependently inhibited by the selective κ antagonist, nor-binaltorphamine (Nor-BNI, 1–20 μg). Whereas both forms of drinking were transiently reduced by the μ-selective antagonist, β-funaltrexamine (β-FNA, 1–20 μg), the μ₁ antagonist, naloxonazine (40 μg) stimulated drinking following hypertonic saline. The δ₁ antagonist, [d-Ala², Leu⁵, Cys⁶]-enkephalin (DALCE, 1–40 μg) significantly reduced drinking following ANGII, but not following hypertonic saline; the δ antagonist, naltrindole failed to exert significant effects. These data indicate that whereas κ opioid binding sites modulate hyperdipsia following hypertonic saline, μ₂, δ₁ and κ opioid binding sites modulate hyperdipsia following ANGII. The μ₁ opioid binding site may normally act to inhibit drinking following hypertonic saline.     

Opioid receptor density changes in Alzheimer amygdala and putamen

Since opioids can influence the release of acetylcholine, substance P and a number of other neurotransmitter that have been implicated in the pathogenesis of Alzheimer's disease (AD), it is of interest to assess opioid receptor levels in AD. We have examined μ, δ and κ opioid receptor binding parameters, binding sensitivity to a GTP analog and distribution in amygdala, frontal cortex and putamen of AD brain. Control brains were matched according to age, sex, post-mortem internal and storage time. K_d values and GTP analog binding sensitivity did not differ in AD and control brains. B_max values for μ ([³H]DAMGE) sites also appeared unaffected by in vitro binding assays. In contrast, κ ([³H]U69593) and δ ([³H]DSLET) opioid receptor levels, were significantly changed. In AD amygdala κ B_max values increased from control levels of 123 ± 12 to 168 ± 13fmol/mg protein, whereas densities of κ and δ sites were decreased from > 94 ± 8 to 48 ± 8 and 102 ± 3.6 to 69 ± 8.5fmol/mg protein, respectively, in putamen. Autoradiography revealed corresponding differences in the distribution of κ opioid receptors. The findings indicated that the κ binding site, which is quantitatively the major opioid receptor class in human brain, undergoes marked changes in AD amygdala and putamen.     

Palatability-induced hyperphagia increases hypothalamic Dynorphin peptide and mRNA levels

Opioid involvement in regulating the intake of highly palatable diets was studied by examining the effect of feeding either a cornstarch-based diet (CHO) or a high fat diet containing sucrose (Fat/Sucrose) on hypothalamic opioid levels. Rats received either CHO ad libitum, Fat/Sucrose ad libitum, Fat/Sucrose pair-fed to the caloric intake of CHO, or Fat/Sucrose at 60% of ad libitum Fat/Sucrose intake. Animals receiving Fat/Sucrose ad libitum consumed more calories and gained more weight than animals receiving CHO (P < 0.001). Relative to CHO, ad libitum intake of Fat/Sucrose elevated proDynorphin mRNA levels in the arcuate and Dynorphin A_1–17 levels in the paraventricular nucleus (PVN) (P < 0.05), but did not affect arcuate mRNA levels of proEnkephalin or proOpiomelanocortin (POMC), or PVN levels of Met-Enkephalin or β-Endorphin. Pair-feeding the Fat/Sucrose diet to the level of intake of the CHO diet resulted in levels of proDynorphin and Dynorphin A_1–17 that were similar in the two diet groups. Pair-feeding Fat/Sucrose reduced mRNA levels of proDynorpin, proEnkephalin and POMC, and Dynorphin A_1–17 levels, relative to ad libitum feeding of Fat/Sucrose. Met-Enkephalin and β-Endorphin were not affected by dietary treatment. Feeding Fat/Sucrose at 60% of ad libitum intake resulted in mRNA levels of proDynorphin, proEnkephalin and POMC, and Dynorphin A_1–17 levels that were similar to those observed in CHO group. Hypothalamic Dynorphin A_1–17 and proDynorphin mRNA levels are stimulated by feeding a highly palatable diet rich in fat and sucrose. The increased synthesis may be due in part to a palatability-induced overconsumption of calories. Caloric restriction of the same diet decreases mRNA levels of proDynorphin, proEnkephalin and POMC, as well as levels of Dynorphin A_1–17.     

Differential effects of μ and κ opioid antagonists on Fos-like immunoreactivity in extended amygdala

It was previously reported that systemic administration of the nonselective opioid antagonist, naltrexone, induces Fos-like immunoreactivity (FLI) within the central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (lateral–dorsal division; BSTLD), nucleus accumbens shell (NACshell) and ventral tegmental area (VTA) of free-feeding rats. These findings suggest that cellular activity in these brain regions is subject to opioid-mediated inhibitory control under basal conditions. Considering the involvement of mesoaccumbens dopamine neurons and components of the ‘extended amygdala' in motivated behavior and reward, it was hypothesized that the induction of c-Fos by naltrexone accounts for the motivational-affective consequences of opioid antagonism. In Experiment 1, naltrexone was administered intracerebroventricularly (i.c.v.; 100 μg) to determine whether results obtained in the prior immunohistochemical studies could be attributed to blockade of opioid receptors in brain as opposed to peripheral tissues that convey visceral sensory inputs to the CeA and BSTLD. Naltrexone produced a marked increase in FLI within the CeA and BSTLD, and a moderate increase in NACshell. In Experiment 2, the κ opioid antagonist, nor-binaltorphimine (Nor-BNI; 20.0 μg, i.c.v.) reproduced the effect of naltrexone in BSTLD and CeA, suggesting that the induction of c-Fos in these two structures is a consequence of κ receptor blockade. The selective μ antagonist, CTAP (2.0 μg, i.c.v.), reproduced the effect of naltrexone in NACshell, suggesting that the induction of c-Fos in this structure is a consequence of μ receptor blockade. The functional implications of these results are discussed in terms of the known functions of these brain regions and opioid receptor types, and the prior observation that chronic food restriction eliminates the FLI induced by naltrexone in CeA and BSTLD. It is suggested that tonic μ opioid-mediated inhibition in NACshell has a predisposing effect on goal–approach behavior in general while κ opioid-mediated inhibition in CeA and BSTLD has a predisposing effect on palatability-driven feeding in particular. Finally, a possible relationship between food restriction-induced suppression of the κ opioid mechanism in CeA/BSTLD, local CRH function, and sensitization of the neural substrate for incentive-motivating effects of abused drugs is discussed.     

The role of opioids in newborn pig fluid percussion brain injury

The present study was designed to characterize the relationship between cerebral opioid concentration, cerebral hemodynamics, and cerebral oxygenation following percussion brain injury in neonatal pigs. Previous research found that opioids represent a significant vasoactive component in the regulation of the neonatal piglet cerebral circulation. Anesthesized newborn (1–5 days old) pigs equipped with a closed cranial window were connected to a percussion device consisting of a saline-filled cylindrical reservoir with a metal pendulum. Brain injury of moderate severity (1.9–2.3 atm.) was produced by allowing the pendulum to strike a piston on the cylinder. Fluid percussion brain injury decreased pial arteriolar diameter (132 ± 5 to 110 ± 5 μm within 10 min). Cerebral blood flow also fell within 10 min of injury and continued to fall progressively for 3 h, resulting in a 46 ± 4% decrease. Within 30 s of brain injury, there was a transient increase in cerebral hemoglobin-O₂ saturation that was reversed to a progressive profound decrease in cerebral hemoglobin-O₂ saturation for the next 3 h, as measured by near infrared spectroscopy. CSF opioid concentrations were increased 10 min after brain injury; dynorphin showed the largest proportional increase (5.8 ± 0.9 fold). The CSF concentration for other opioids continued to increase over 180 min while the dynorphin concentration progressively decreased with time. In naloxone (1 mg/kg i.v.) pretreated piglets, the brain injury induced decrease in arteriolar diameter was attenuated (129 ± 5 to 121 ± 5 μm within 10 min). Similarly, the decrease in regional cerebral blood flow and cerebral hemoglobin-O₂ saturation observed following brain injury were also blunted by naloxone. These data show that CSF opioid concentrations increase following brain injury and that the time course and relative increase in CSF concentration vary from opioid to opioid. These data also indicate that in the immature animal, opioids contribute to arteriolar constriction, decreased cerebral blood flow, decreased cerebral oxygenation, and could play a role in causing ischemia after brain injury.     

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.     

μ-, δ-, κ- and ε-Opioid receptor modulation of the hypothalamic-pituitary-adrenocortical (HPA) axis: subchronic tolerance studies of endogenous opioid peptides

In opiate-naive rats, the endogenous opioid peptides, β-endorphin, dynorphin(1–13) and Met---Enk---Arg---Phe (MEAP) and the synthetic enkephalin analogue -Ala²- -Leu⁵-Enk (DADLE) potently stimulated plasma corticosterone in a dose-dependent, naloxone-reversible manner. To characterize their in vivo affinities, the effects of these peptides on plasma corticosterone release were tested in rats made tolerant to morphine, U50488H, DADLE/morphine or β-endorphin. These cross-tolerance studies showed that dynorphin and MEAP exerted their action on plasma corticosterone release at κ-opioid receptors. The action of DADLE occurred at δ-opioid receptors, while the action of β-endorphin occurred principally at another receptor site. These results indicate that there is independent modulation of the hypothalamic-pituitary-adrenal axis by endogenous opioid peptides at μ-, δ- and κ-opioid receptors. In addition, there may be modulation by β-endorphin at a separate site that we suggest could be a central ε-receptor site. This cross-tolerance paradigm, using a neuroendocrine model, provides in vivo evidence for the action of centrally active endogenous opioid peptides at multiple and independent opioid receptors.     

Pharmacological characterization of buprenorphine, a mixed agonist–antagonist with κ3 analgesia

Buprenorphine is a mixed opioid agonist/antagonist analgesic. This study was designed to determine the role of opioid receptor subtypes, especially κ₃, in buprenorphine-induced analgesia in mice. Buprenorphine, when injected systemically, revealed a potent analgesic effect by tailflick assay, with a biphasic dose–response curve, which was reversed by naloxone. The presence of analgesic cross-tolerance between buprenorphine and naloxone benzoylhydrazone (NalBzoH) and morphine indicated a role for κ₃ and μ receptor subtype in buprenorphine analgesia. Additional studies with selective opioid antagonists indicated κ₁ mechanisms of action. We did not detect any involvement of the δ receptor subtype. Low doses of buprenorphine antagonized morphine analgesia, while high doses of buprenorphine coadministered with morphine elicited increasing analgesia in a dose-dependent manner. These findings suggest that buprenorphine elicits analgesia through an interaction with κ₃ receptors and to a lesser extent with κ₁ as well as its activity as partial μ receptor agonist.     

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg⁶ and to a lesser extent to Leu-enkephalin. Five day treatment with 10 μM morphine increased the conversion to Leu-enkephalin-Arg⁶ by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B>-neoendorphin>>dynorphin A. The K_m for the generation of Leu-enkephalin-Arg⁶ from dynorphin B was 40 μM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1–13)>A(2–13)>A(1–17)>A(2–17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for κ-opioid receptors) and enkephalin peptides (selective for δ-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

Morphine-induced place preference in the CXBK mouse: characteristics of μ opioid receptor subtypes

The role of μ opioid receptor subtypes, μ₁ and μ₂, in morphine-conditioned place preference was examined using ddY and μ₁ opioid receptor-deficient CXBK mice. In ddY mice, the μ receptor agonist morphine caused a dose-related preference for the drug-associated place, but the κ agonist U-50,488H produced a dose-related place aversion. These results demonstrated that the mouse is available for place preference conditioning using opioids. Under this condition, the influence of pretreatment with selective μ₁ opioid receptor antagonist naloxonazine morphine-induced place preference was investigated in ddY mice. Although pretreatment with the selective μ₁ antagonist naloxonazine (35 mg/kg, s.c.) did not modify the morphine-induced place preference, pretreatment with the selective μ antagonist β-funaltrexamine (β-FNA 10 mg/kg, s.c.) eliminated the appetitive effect of morphine. Furthermore, morphine (1–5 mg/kg, s.c.) produced a dose-related preference for the drug-associated place in CXBK mice. These findings suggest that the morphine-induced conditioned place preference may be mediated by naloxonazine-insensitive sites (μ₂ opioid receptors). In addition, chronic infusion of the dopamine D₁ antagonist SCH23390 (1.0 mg/kg/day) during the conditioning sessions eliminated the morphine-induced place preference in CXBK mice. Similarly, morphine combined with naloxonazine failed to produce the place preference in ddY mice chronically treated with SCH23390. The blocking effect of SCH23390 on the morphine-conditioned place preference suggests that μ₂ receptors may regulate the dopaminergic system, especially dopamine D₁ receptors, and are also involved in the reinforcing effects of morphine.     

Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line

Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that ³H-dynorphinA-(1-13)-NH₂ associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a K_d of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with ³H-dynorphinA-(1-13)-NH₂ for that site with IC₅₀s ranging from 200 nM to 2 μM. ACTH(1-39) also competes with ³H-dynorphinA-(1-13)-NH₂ for the site with an IC₅₀ of about 300 nM. DynorphinA-(2-17) at μM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.     

Chronic food restriction alters μ and κ opioid receptor binding in the parabrachial nucleus of the rat: a quantitative autoradiographic study

Using quantitative autoradiography, it was previously observed that chronic food restriction alters μ and κ receptor binding in several regions of the rat forebrain. The present autoradiographic study was designed to investigate whether food restriction affects regional μ and κ binding in the brainstem. [³H]DAGO (It,) and μ/δ blocked [³H]BMZ (κ) binding were analyzed in 21 brainstem regions. A signficant decrease in μ binding was observed in the external lateral and external medial subnuclei of the parabrachial nucleus while a significant increase in κ binding was observed in the external lateral subnucleus. The possible functional significance of these changes is discussed.     

Excitatory amino acid receptor subtype agonists induce feeding in the nucleus accumbens shell in rats: opioid antagonist actions and interactions with μ-opioid agonists

Administration of μ-opioid receptor subtype agonists into the nucleus accumbens shell elicits feeding which is dependent upon the normal function of μ-, δ- and κ-opioid receptors, D₁ dopamine receptors and GABA_B receptors in the nucleus accumbens shell for its full expression. Whereas the AMPA antagonist, DNQX administered into the nucleus accumbens shell elicits a transient, though intense feeding response, feeding is elicited by excitatory amino acid agonists administered into the lateral hypothalamus. The present study examined whether excitatory amino acid agonists elicited feeding following administration into the nucleus accumbens shell of rats, whether such feeding responses were altered by opioid antagonist pretreatment, and whether such feeding responses interacted with feeding elicited by μ-opioid agonists. Both AMPA (0.25–0.5 μg) and NMDA (1 μg) in the nucleus accumbens shell significantly and dose-dependently increased food intake over 4 h. Both feeding responses were blocked by naltrexone pretreatment in the nucleus accumbens shell. The μ-opioid agonist, [D-Ala²,NMe-Phe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell significantly increased food intake which was significantly enhanced by AMPA cotreatment. This enhanced feeding response was in turn blocked by pretreatment with either general or μ-selective opioid antagonists. In contrast, cotreatment of NMDA and the μ-opioid agonist in the nucleus accumbens shell elicited feeding which was significantly less than that elicited by either treatment alone. These data indicate the presence of important interactions between excitatory amino acid receptors and μ-opioid receptors in the nucleus accumbens shell in mediating feeding responses in nondeprived, ad libitum-fed rats.     

Dynorphin A-(1–13) attenuates basal forebrain-lesion-induced amnesia in rats

The effects of dynorphin A-(1–13), an endogenous κ opioid agonist, on basal forebrain (BF)-lesion-induced amnesia in rats were investigated using step-through-type passive avoidance task. The BF was lesioned by injecting the cholinergic neurotoxin ibotenic acid (6 μg/side). The number of rats achieving the cut-off time (600 s) of step-through latency (STL) in BF-lesioned group significantly decreased as compared with that in sham-operated group. Dynorphin A-(1–13) (0.3 μg) significantly increased the number of rats achieving the cut-off time of STL in BF-lesioned rats. These results suggest that dynorphins play an improving role in the impairment of memory processes in BF-lesioned rats.     

The effect of U-50,488H tolerance-dependence and abstinence on the levels of dynorphin (1–13) in brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a κ-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1–13), whereas the heart had the lowest level. The levels of dynorphin (1–13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1–13) were elevated only in the midbrain. The levels of dynorphin (1–13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1–13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1–13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the μ- and κ-opiate induced tolerance-dependence and abstinence processes.