Ellen M. Unterwald

Retention of Supraspinal Delta-like Analgesia and Loss of Morphine Tolerance in δ Opioid Receptor Knockout Mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.

Retention of heroin and morphine–6β–glucuronide analgesia in a new line of mice lacking exon 1 of MOR–1

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR–1 gene. Although morphine–6β–glucuronide (M6G), heroin and 6–acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR–1. Mice homozygous for either MOR–1 mutation were insensitive to morphine. Heroin, 6–acetylmorphine and M6G still elicited analgesia in the exon–1 MOR–1 mutant, which also showed specific M6G binding, whereas M6G and 6–acetylmorphine were inactive in the exon–2 MOR–1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors.

The present study investigated the regulation of opioid receptors following chronic repeated cocaine administration. Male Fischer rats were injected with saline or cocaine, 45 mg kg-1 day-1, three times daily at 1 h intervals for 14 days. Using quantitative in vitro autoradiography, the density of kappa receptors was significantly higher in the rostral cingulate cortex, rostral caudate putamen, caudal olfactory tubercle, and ventral tegmental area following cocaine administration. No changes in delta receptor number were detected in any brain region examined. Significant mu receptor upregulation was found in the rostral cingulate cortex, nucleus accumbens, rostral caudate putamen, and basolateral amygdala nucleus of the cocaine-treated animals. These results suggest that enhanced dopaminergic activity can regulate mu and kappa opioid receptors.

Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR–1 gene. Although morphine–6β–glucuronide (M6G), heroin and 6–acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR–1. Mice homozygous for either MOR–1 mutation were insensitive to morphine. Heroin, 6–acetylmorphine and M6G still elicited analgesia in the exon–1 MOR–1 mutant, which also showed specific M6G binding, whereas M6G and 6–acetylmorphine were inactive in the exon–2 MOR–1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Chronic cocaine alters brain mu opioid receptors

The possibility that dopamine may modulate the expression of opioid receptors was investigated by determining the effects of chronic cocaine administration on the density of mu opioid receptors. Quantitative in vitro autoradiography with the highly selective mu opioid ligand [3H]DAMGO was used to measure and localize changes in mu opioid receptors in the brains of rats administered cocaine or saline three times daily for 14 days. Significant increases in [3H]DAMGO binding were measured in areas of the cingulate cortex, nucleus accumbens, caudate putamen, and basolateral amygdaloid nucleus of the cocaine-treated animals. These results demonstrate that mu opioid receptors undergo upregulation in response to chronic cocaine exposure and suggest that dopamine activity can regulate the expression of mu opioid receptors.

Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay

Quantitative solution hybridization assays were used to determine the picogram amounts of preproenkephalin mRNA (PPenk mRNA) and the microgram quanities of total rat RNA in extracts of eight brain regions from rats which had received three daily intraperitoneal injections of cocaine (10 or 30 mg/kg/day) or saline for 14 days. The young adult male Fischer rats were sacrificed 30 min after the final injection. The highest density of PPenk mRNA (pg PPenk mRNA/micrograms total cellular RNA) was found in extracts of striatum (34.08 +/- 1.79 pg/micrograms for 11 saline-treated rats), followed by extracts of nucleus accumbens (10.08 +/- 0.81 pg/micrograms), and extracts of hypothalamus (2.99 +/- 0.31 pg/micrograms). Extracts of frontal cortex (1.78 +/- 0.24 pg/micrograms), pituitary (1.39 +/- 0.08 pg/micrograms), central grey (1.31 +/- 0.16 pg/micrograms), and cerebellum (1.24 +/- 0.09 pg/micrograms) had intermediate values. Extracts of hippocampus (0.53 +/- 0.03 pg/micrograms) had the lowest density. No significant differences were found among the treatment groups in any brain area investigated. Therefore, chronic cocaine treatment as administered in this protocol did not alter expression of the gene encoding proenkephalin.

Delta opioid receptor ligands modulate anxiety-like behaviors in the rat

The role of the delta opioid receptor in regulating anxiety‐like behavior in male Sprague–Dawley rats was examined. Using an elevated plus maze, the effects of the selective delta opioid receptor antagonist naltrindole (1 or 5 mg kg−1) and agonist SNC80 (1, 5 or 20 mg kg−1) on anxiety‐like behavior were measured. Anxiety was also measured following administration of diazepam (3 mg kg−1) and amphetamine (1 mg kg−1) and compared to the effects of SNC80. Locomotor activity following administration of naltrindole, SNC80, diazepam, and amphetamine was measured. Finally, the defensive burying paradigm was used to confirm the findings from the elevated plus maze. Results demonstrated that SNC80 produced dose‐dependent anxiolytic effects similar to that of the classical antianxiety agent, diazepam. Administration of naltrindole caused anxiogenic behavior in rats further supporting the involvement of the delta opioid receptor system in regulating anxiety. Naltrindole also blocked the anxiolytic effects of SNC80. Amphetamine had no effect on anxiety‐like behavior. SNC80 induced hyperactivity similar to amphetamine at the doses tested, while naltrindole and diazepam did not significantly affect locomotor activity. Although SNC80 can increase locomotor activity, control experiments reported herein indicate that hyperlocomotion is not sufficient to produce an anxiolytic response on the elevated plus maze. Together with the results from the defensive burying paradigm, this suggests that the effects of SNC80 on reducing anxiety are independent of its effects on locomotion. Collectively these data show that the delta opioid receptor system can regulate anxiety‐like behavior in an anxiolytic (agonist) and anxiogenic (antagonist) manner.

The frequency of cocaine administration impacts cocaine-induced receptor alterations

The present study investigated the impact of dosing schedule on cocaine-induced receptor alterations. Rats were injected with 30 mg/kg per day of cocaine given either as a single injection or in two equally divided doses for 14 days. The effects of these two dosing regimens were compared with our previous findings following administration of cocaine three times daily at 1-h intervals. Using receptor autoradiography, twice daily injections of cocaine produced an upregulation of mu opioid receptors in the rostral nucleus accumbens, rostral caudate putamen, and layer I of the rostral cingulate cortex, whereas single daily injections resulted in a significant increase in the nucleus accumbens only. Only small insignificant increases in kappa opioid receptor densities were found following either once or twice daily cocaine injections, whereas three daily injections produced an increase in kappa receptor density in the cingulate cortex, nucleus accumbens, and caudate putamen. Increased dopamine D1 receptor binding was found in the nucleus accumbens and olfactory tubercle following twice daily cocaine injections, but not after single daily injections of the same total daily dose. These results demonstrate that the same total daily dose of cocaine administered in multiple small injections produces a greater effect on receptor regulation than a single larger injection. This suggests that the interval between cocaine injections is an important variable when studying the effects of cocaine on neurochemistry.

Withdrawal from chronic administration of cocaine decreases delta opioid receptor signaling and increases anxiety- and depression-like behaviors in the rat

Chronic administration of cocaine has been shown to attenuate the functional capacity of delta opioid receptors to inhibit adenylyl cyclase activity. Abuse and withdrawal from cocaine in humans is associated with increases in anxiety and depression. Since recent research supports the role of delta opioid receptors in anxiety- and depression-like behaviors in rodents, we hypothesized that functional desensitization of delta opioid receptors contributes to anxiety- and depression-like behavioral phenotypes following short-term withdrawal from chronic administration of cocaine. To test this hypothesis, delta opioid receptor signaling and behaviors were evaluated 24h after 14days of binge-pattern cocaine administration (15mg/kg three times daily at 1h intervals) in male Sprague-Dawley rats. Results showed that the inhibition of adenylyl cyclase by delta opioid receptor agonists was attenuated in the frontal cortex, nucleus accumbens and caudate putamen 24h after cessation of cocaine administration. One day withdrawal from chronic administration of cocaine resulted in increased anxiety- and depression-like behaviors as measured by the elevated plus maze and the forced swim test respectively, and no change in locomotor activity. The anxiety- and depression-like behaviors were dose-dependently reduced by acute administration of the selective delta opioid receptor agonist, SNC80. These results demonstrate that early withdrawal from cocaine resulted in increased anxiety and depression, which accompanies the desensitization of delta opioid receptor function. Furthermore, cocaine-induced anxiety- and depression-like behaviors were reversible by the delta opioid receptor agonist SNC80.

Chronic opioid antagonist administration upregulates mu opioid receptor binding without altering mu opioid receptor mRNA levels

Chronic administration of opioid antagonists has been shown to increase radioligand binding to brain opioid receptors. The present study was conducted to determine whether chronic exposure to the opioid antagonist naltrexone would similarly increase mu opioid receptor gene expression as measured by mRNA levels. Male Sprague-Dawley rats were administered naltrexone, 7-8 mg/kg/day, or saline by osmotic minipumps for 7 days. As expected, the density of mu opioid receptor binding sites was significantly higher in the brains of animals treated chronically with naltrexone as compared with saline-treated control animals. However, mu opioid receptor mRNA content determined by a solution hybridization RNase protection assay was not significantly altered in any brain region investigated. These results indicate that the upregulation of mu opioid receptors as measured by radioligand binding is not accompanied by increased levels of mu receptor mRNA.