Comparison of naltrexone and quaternary naltrexone after systemic and intracerebroventricular administration in pigeons.

The behavioral effects of naltrexone and quaternary naltrexone were evaluated in two groups of pigeons. One group responded under a fixed-ratio schedule of food reinforcement and was trained to discriminate between 3.2 mg/kg morphine (i.m.) and saline. Drug-appropriate responding occurred in a dose-related manner to morphine, given intramuscularly and intraventricularly. When administered intraventricularly morphine was 50 times more potent as a discriminative stimulus and 50-100 times more potent at suppressing responding. Naltrexone, given intraventricularly and intramuscularly, attenuated the discriminative stimulus effects of morphine. Quaternary naltrexone was more potent at suppressing responding when administered intraventricularly but it failed to attenuate the discriminative stimulus effects of morphine. A second group of pigeons, responding under a variable-interval schedule of food reinforcement, was treated with 100.0 mg/kg/day of morphine. Naltrexone and quaternary naltrexone suppressed responding by both routes of administration. Naltrexone was approximately equipotent when given intramuscularly or intraventricularly, and the doses that suppressed responding were 50-500 times smaller than doses required to suppress responding in untreated pigeons. Although quaternary naltrexone was 1800 times more potent when given intraventricularly, the doses necessary to suppress responding by each route were the same as doses required in untreated pigeons. These results extend the conditions under which a quaternary derivative of naltrexone failed to display antagonist activity in the pigeon. The utility of this compound for characterizing central and peripheral mechanisms of action has not been established in different species and testing conditions and, therefore, appears to be appropriate only under conditions in which it is evaluated after systemic, as well as central, administration.

Tolerance and dependence after continuous morphine infusion from osmotic pumps measured by operant responding in rats.

Food-deprived rats were trained to lever-press on a fixed interval 3-min schedule of food presentation. Using a cumulative dosing procedure, morphine and naltrexone were tested weekly for effects on rates of responding. A separate group of subjects was tested weekly for morphine analgesia using the tail-flick assay. Chronic morphine infusion (10 mg/kg/day, SC, from a 4-week osmotic pump) induced tolerance to the rate-decreasing and analgesic effects of morphine as demonstrated by a more than 4-fold increase in the morphine ED50s for both of these effects, relative to those of predependent rats. Physical dependence was evidenced in the operant procedure by increased sensitivity to the rate-decreasing effects of the opioid antagonist, naltrexone, such that the ED50 for naltrexone was 5000 times lower than it was in predependent animals. In addition, marked weight loss was observed over the 2-h naltrexone test session. The degree of tolerance was a function of both the length of chronic administration and of the maintenance dose and was of a similar magnitude for operant responding and analgesia. Further, the degree of tolerance and dependence was comparable to that observed in other studies that used different methods of chronic drug administration. Thus, osmotic pumps provide a convenient and effective alternative for chronic drug administration in behavioral studies.

Conditioned locomotor stimulant effects of cocaine in rats do not result from interference with habituation.

RATIONALE: Classical conditioning has been proposed to account for the hyperactivity observed in drug-free rats when placed in an environment previously paired with cocaine administration. However, an alternative explanation is that hyperactivity results from an inability of rats to habituate to the environment under the influence of cocaine. OBJECTIVES: In this study, preconditioning exposure to the test environment was increased from one session (standard procedure) to seven (modified procedure) to test the "antihabituation" hypothesis. METHODS: After preconditioning exposure, six conditioning sessions took place over a 10-day to 13-day period. Paired rats received 10 mg/kg cocaine i.p. prior to activity sessions and saline i.p. upon return to the colony room. Unpaired rats received saline prior to and cocaine after activity sessions. Time-off rats were withheld from the activity boxes, but were subject to all other procedures during conditioning. On the test day, all rats received saline prior to activity sessions. RESULTS: In the standard procedure, paired rats exhibited significantly greater activity than unpaired rats on the test day, consistent with previous reports. In the modified procedure, mean activity (all rats) decreased between the first and last preconditioning sessions. Still, the paired group exhibited greater activity than the unpaired group on the test day, suggesting that a conditioned stimulant effect developed in habituated rats. Activity in the time-off group did not significantly differ from the unpaired group demonstrating the habituation had not dissipated over this time period. CONCLUSIONS: These results support the conclusion that hyperactivity observed on the test day was not a result of antihabituation effects of cocaine.

Intracerebroventricular treatment with an antisense oligodeoxynucleotide to kappa-opioid receptors inhibited kappa-agonist-induced analgesia in rats.

In vivo treatment with an antisense (AS) phosphorothioate oligodeoxynucleotide (oligo) to the rat kappa-opioid receptor selectively inhibited kappa-mediated analgesia in the rat cold-water tail-flick test. Intracerebroventricular (i.c.v.) AS oligo significantly inhibited the analgesic effect of i.c.v. spiradoline, but not that of mu- or delta-opioid agonists. The dose-effect curve for s.c. spiradoline was shifted to the right after AS, but not missense or sense oligo treatment. Thus, AS oligos provide another technique with which to selectively manipulate opioid receptors and further support the role of non-mu opioid receptors in mediating analgesia in rats.

Naltrexone-sensitizing effects of centrally administered morphine and opioid peptides.

Rats were trained to lever-press on a multiple-trial, fixed-interval, 3-min schedule of food-reinforcement; each trial consisted of a 10-min time-out and a 9.5-min response period. Naltrexone, injected s.c. prior to each trial, reduced response rates in a dose-related fashion, with an ED50 of 21 mg/kg. Four-hour pretreatment with i.c.v. morphine (3.0-30 micrograms) produced leftward shifts of the naltrexone dose-effect curve of up to 3 orders of magnitude. The selective mu agonist, [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO; 0.1-0.3 microgram i.c.v.), sensitized animals to the rate-decreasing effects of naltrexone by more than 2 orders of magnitude. Pretreatment with the selective kappa agonist, dynorphin A-(1-17) (30 micrograms i.c.v.) or the selective delta agonist, [D-Pen2, D-Pen5]enkephalin (DPDPE; 30-100 micrograms i.c.v.) induced moderate sensitization with leftward shifts of 1.0-1.5 log units. Thus, the naltrexone-sensitizing effect of acute opioid pretreatment is centrally mediated, consistent with the hypothesis that the phenomenon is related to the initiation of opioid physical dependence. Further, the effect appears to be mediated predominantly by mu-opioid receptors, because mu agonists consistently produced the largest sensitization to naltrexone.

An antisense oligodeoxynucleotide to mu-opioid receptors inhibits mu-opioid receptor agonist-induced analgesia in rats.

We examined effects of an antisense oligodeoxynucleotide against the mu-opioid receptor on mu-opioid receptor agonist-induced antinociception in the cold water (-3 degrees C) tail-flick test in rats. Rats were injected intracerebroventricularly (i.c.v.) with an antisense, sense or missense oligodeoxynucleotide or artificial cerebrospinal fluid on days 1, 3 and 5. On day 6, antinociceptive effects of opioid agonists were tested. Compared to the artificial cerebrospinal fluid treatment, the cumulative dose-effect curve for subcutaneous (s.c.) morphine was shifted to the right by the antisense oligodeoxynucleotide, but not by the missense oligodeoxynucleotide or the sense oligodeoxynucleotide treatment. Antisense oligodeoxynucleotide treatment reduced the analgesic effect of the mu-opioid receptor agonist PL017 ([N-MePhe3,D-Pro4]morphiceptin), but not the delta-opioid receptor agonist BW373U86 ((+/-)-4-((a-R*)-a-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- hydroxybenzyl)-N,N-diethyl-benzamide) or the kappa-opioid receptor agonist spiradoline ((+/-)-(5a,7a,8b)-3,4-dichloro-N-methyl-N-[7-(1- pyrrolidinyl)-1-(oxaspiro-[4,5]dec-8-yl]benzeneacetamide monohydrochloride). The drugs were given by i.c.v. injection. These findings indicate that i.c.v. administration of a mu antisense oligodeoxynucleotide specifically blocks mu-, but not delta- or kappa-opioid receptor-mediated analgesia in the rat cold water tail-flick test.

Receptor selectivity of icv morphine in the rat cold water tail-flick test.

The cold water tail-flick test in the rat is somewhat unique in that it is sensitive to the analgesic effects of delta- and kappa- in addition to mu-opioid agonists. The present study was designed to test whether a component of morphine-induced analgesia in this test might be mediated by delta- or kappa-opioid receptors. Morphine was administered icv in combination with the non-selective opioid antagonist naloxone (NLX), as well as the mu-, delta- and kappa-selective antagonists, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr (CTAP), naltrindole (NTI) and norbinaltorphimine (norBNI), respectively. Morphine induced analgesia in a dose related manner. Administration of NLX (1-10 micrograms) or CTAP (1 microgram) antagonized morphine in a competitive fashion. Neither NTI (1-10 micrograms) nor norBNI (0.1 microgram) had any effect on the morphine dose-effect curve. Thus, morphine appeared to be a selective mu agonist in the cold water tail-flick test, at least by the icv route.

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats.

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.

Pyrogenic doses of intracerebroventricular interleukin-1 did not induce analgesia in the rat hot-plate or cold-water tail-flick tests.

There are a few reports in the literature that cytokines can induce analgesia (5, 6, 18). The present study sought to characterize the analgesic effects of intracerebroventricularly (icv) administered interleukin-1 (IL-1) and interferon-alpha (IFN-alpha) in rats. In the cold-water tail-flick test (CWT), latency to tail withdrawal from a -3 degrees C liquid was timed; in the hot-plate test (HP), latency to a rear paw lick or a jump from a 55 degrees C surface was measured. In some experiments, core body temperature was also monitored with a rectal thermistor. In the CWT, human recombinant (hr) IFN-alpha induced a small, statistically significant effect at just one dose (15,000 U icv), but no dose of hr-IL-1 alpha (250-1000 U icv) or hr-IL-1 beta (125-2000 U icv) induced a significant effect at any time point. On the other hand, dose-related increases in body temperature were observed after icv injection of both IL-1 alpha and IL-1 beta. The largest hyperthermic effect was a 1.7 (+/- 0.15) degrees C rise 120 min after administration of 1000 U IL-1 beta. In a second analgesic assay, the HP, IL-1 beta was ineffective as well. Since IL-1 alone did not induce analgesia, we tested its capacity to potentiate morphine analgesia. Morphine (5.0 and 10 micrograms, icv) induced analgesia in the CWT (32.7 and 61.8% maximum analgesia, respectively); however, there was no significant effect of IL-1 beta on morphine-induced analgesia. In summary, we failed to find an analgesic effect of IL-1, alone or in combination with morphine, at doses which clearly had a physiological effect; this is in contrast to the reports cited above.

Effects of receptor-selective opioids on operant behavior in morphine-treated and untreated rats.

Selective cross-tolerance is often used as evidence to differentiate opioid receptor subtypes. We used this strategy to study operant behavioral effects of the opioid peptides, [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAGO), [D-Pen2.5]-enkephalin (DPDPE) and dynorphin, agonists highly selective for mu, delta, and kappa receptors, respectively. Food-deprived rats were trained to lever-press on a fixed-interval 3-min schedule of food-reinforcement. Time-effect and dose-effect curves were generated for each of the peptides, as well as for morphine, administered ICV, 5 min prior to the 1-h operant session. Experiments were performed on untreated subjects and on subjects receiving chronic infusion of morphine (10 mg/kg/day) from osmotic pumps. In untreated animals, morphine and the mu-selective peptide, DAGO, induced relatively long-lasting dose-related decreases in responding, whereas the non-mu agonists, DPDPE and dynorphin, induced only transient effects: response rates increased at low doses and decreased at high doses. Animals receiving chronic morphine infusion were tolerant to the rate-decreasing effects of morphine and DAGO; ED50s increased by factors of 8 and 6, respectively. There was some evidence of cross-tolerance to DPDPE and of sensitization to dynorphin in the morphine-maintained animals.

Prepulse inhibition of the acoustic startle response in cocaine-withdrawn rats.

Prepulse inhibition (PPI) of startle is a sensorimotor gating task in which a low-intensity acoustic stimulus presented prior to a high-intensity, startle-eliciting stimulus can attenuate the acoustic startle response (ASR). Previous studies on startle reactivity in cocaine-withdrawn rats have found minimal changes; the present study extends this work to the gating of ASR. In Experiment 1, rats were injected daily with either saline or cocaine (30 mg/kg i.p.) for 2 weeks. ASR and PPI were measured prior to, and at 3- and 14-day withdrawal from, the chronic treatment. No effect of cocaine treatment was found on either measure. In Experiment 2, treatment was extended to 8 weeks, and an earlier withdrawal time point (1 day) was added. Rats treated with cocaine for 8 weeks exhibited lower startle reactivity during withdrawal compared with saline-treated controls. PPI did not differ between treatment groups. Thus, extended chronic treatment with cocaine rendered significant effects on startle responsivity. Further, this finding mirrors the blunted ASR exhibited in chronic cocaine users [Neuropsychopharmacology 22 (2000) 89.].

Intracerebroventricular drug administration in pigeons.

For many procedures used in behavioral pharmacology, the intracerebroventricular (ICV) route of drug administration is infrequently used due, in part, to the lack of a reliable technique for determining cannula patency in vivo. This study describes an in vivo technique for assessing ICV cannula patency in pigeons. The technique was applied in an experiment designed to evaluate several drugs, which are presumed to differ in the extent to which they enter the central nervous system, for their rate-suppressing effects in pigeons trained to peck a key on a fixed-ratio 20 schedule of food reinforcement. The opioid agonist morphine and antagonist quaternary naltrexone were 100 and 280 times more potent, respectively, in suppressing responding when administered ICV, as compared to systemic administration. Tertiary naltrexone was approximately equipotent as an antagonist of morphine's rate-suppressing effects when administered ICV or systemically. Quaternary naltrexone did not antagonize morphine by either route of administration. The utility of this in vivo cannula verification technique is discussed, as well as the limitations of comparisons between systemically-administered tertiary and quaternary derivatives.

Pharmacologic characterization of the sensitization to the rate-decreasing effects of naltrexone induced by acute opioid pretreatment in rats.

The pharmacologic specificity of the sensitization to naltrexone induced by acute opioid pretreatment was studied in rats trained to lever-press on a multiple-trial, fixed-interval 3-min schedule of food reinforcement. Cumulative doses of naltrexone were given until responding was suppressed; control naltrexone ED50 values for decreasing response rates ranged from 5.0 to 22 mg/kg. Agonists were administered 4 hr before naltrexone challenge. Pretreatment with morphine (10 mg/kg) initially produced a 4-fold shift to the left of the naltrexone dose-effect curve, but after repeated weekly testing with various agonists, produced a 1700-fold shift. Pretreatment with other millimicrons agonists (i.e., 0.3 mg/kg of levorphanol, 0.06 mg/kg of fentanyl and 3.0 mg/kg of methadone) produced similarly large (100- to 250-fold) increases in sensitivity to the rate-decreasing effects of naltrexone. On the other hand, pretreatment with agonists selective for kappa (1.0 mg/kg of ethylketocyclazocine and 3.0 mg/kg of U-50,488) or sigma (10 mg/kg of [+]-N-allylnormetazocine) receptors, produced smaller (10-fold) changes in sensitization to naltrexone. Neither dextrorphan (3.0 mg/kg) nor pentobarbital (18 mg/kg) pretreatment altered sensitivity to naltrexone. Thus, the sensitization to naltrexone induced by acute opioid pretreatment was a stereoselective, opioid-specific effect, and appeared to be mediated primarily by a mu-opioid mechanism. With repeated testing, the effect of acute morphine pretreatment was comparable to that reported after chronic administration, thus supporting the hypothesis that this phenomenon reflects receptor-mediated changes that underlie the state of opioid physical dependence.

Assessment of relative intrinsic activity of mu-opioid analgesics in vivo by using beta-funaltrexamine.

Morphine is the prototypic mu-opioid analgesic; however, in certain situations in vitro, morphine behaves as a partial agonist. To assess the relative intrinsic activity of morphine and three other mu-opioid analgesics in vivo, beta-funaltrexamine (beta-FNA), an irreversible antagonist selective for the mu receptor, was used to reduce the effective receptor reserve. By using a stereotaxic device, 1.25 to 20 micrograms of beta-FNA was infused into the lateral ventricle of rats. Twenty-four hours later, animals were tested in the tail-flick assay with cumulative doses of morphine, levorphanol, methadone or fentanyl. Pretreatment with 2.5 micrograms of beta-FNA induced parallel rightward shifts of both the morphine and levorphanol dose-effect curves and 5.0 micrograms of beta-FNA reduced the maximum analgesic effect of these agonists. Methadone surmounted the antagonism of 5.0 micrograms of beta-FNA; 10 micrograms was required to reduce the maximum analgesic effect of methadone. Fentanyl overcame the blockade induced by both 5.0 and 10 micrograms of beta-FNA. Only with a pretreatment dose of 20 micrograms of beta-FNA was the maximum analgesic effect of fentanyl reduced. Thus, when a certain proportion of mu receptors is inactivated, i.e., with 5.0 micrograms of beta-FNA, fentanyl and methadone have the capacity to surmount the blockade, whereas morphine and levorphanol do not. This suggests that fentanyl and methadone have higher intrinsic efficacies than do morphine and levorphanol. Thus, a strategy used widely in vitro was applied successfully in vivo to assess relative intrinsic activities of a series of mu-opioid agonists.

Effect of intracerebroventricular beta-funaltrexamine on mu opioid receptors in the rat brain: consideration of binding condition.

Effects of 24 h pretreatment with intracerebroventricular (icv) beta-funaltrexamine (beta-FNA) on brain opioid receptor binding in rats were examined under various conditions. Agonist binding to mu and delta opioid receptors (with [3H][[cap]dAla2,MePhe4,Gly-ol5]enkephalin (DAMGO)[3H][D-Pen2, D-Pen5]enkephalin (DPDPE), respectively) was performed under three different conditions: i) pretreatment of membranes with GDP and Na+ and binding in the presence of Mg++ in Tris-HCI buffer containing EGTA and leupeptin for 1.5 to 3 h; ii) binding in Tris-HCI buffer containing bacitracin, leupeptin, chymostatin and bestatin for 3 to 4 h; iii) binding in Tris-HCI buffer containing EGTA and leupeptin for 45 min. Condition i was shown to convert opioid receptors to a high affinity state for agonists. beta-FNA (2, 6 or 20 nmol) significantly reduced 1 nM [3H]DAMGO binding in the whole brain with i but not with ii. With iii, 20 nmol beta-FNA reduced [3H]DAMGO binding, but not 2 or 6 nmol. Saturation experiments with i showed that the reduction in [3H]DAMGO binding after 6 or 20 nmol beta-FNA was due to a decrease in Bmax and an increase in KD. For delta binding, there was no significant change in [3H]DPDPE (2 nM) binding with i after 2, 6 or 20 nmol beta-FNA. Thus, under i, icv beta-FNA reduced [3H]DAMGO binding significantly without affecting [3H]DPDPE binding. In addition, mu binding was also conducted with 1 nM [3H]naloxone under three different conditions: iv) in the presence of Na+ and GDP; v), in the presence of Na+, Gpp(NH)p and Mg++; vi) in the presence of Na+. Both iv and v were shown to shift opioid receptors to a low affinity state for agonists. beta-FNA (20 nmol) significantly decreased 1 nM [3H]naloxone binding under each of the three conditions. Competitive inhibition of 1 nM [3H]naloxone binding by DAMGO in the presence of Na+ and GDP showed that receptors existed in a single low affinity state for DAMGO, and that icv beta-FNA caused a reduction in Bmax without affecting the KD of DAMGO. In summary, when all the receptors were converted to a high agonist affinity state i or a low agonist affinity state iv, the changes in mu binding induced by beta-FNA could be revealed with agonist binding. Additionally, changes in mu binding induced by beta-FNA could be detected with [3H]naloxone, which always displayed high affinity regardless of agonist affinity states, under each of the three conditions (iv, v and vi).

Isobolographic superadditivity between delta and mu opioid agonists in the rat depends on the ratio of compounds, the mu agonist and the analgesic assay used.

The present study was designed to test rigorously, using isobolographic analysis, whether there was a potentiative interaction between delta and mu agonists administered i.c.v. to rats. Factors such as the specific fixed ratio of compounds, the analgesic assay and the mu agonist were varied to determine the generality of the results. Male rats were implanted with i.c.v. cannulas and were tested in the cold (-3 degrees C) or hot (+50 degrees C) water tail-flick test. Full dose-effect curves were generated for the mu agonists, morphine and [N-MePhe3,D-Pro4]morphiceptin and the delta-selective agonist, DPDPE. Each agonist induced dose-related analgesia in the cold water test (ED50 values were 12, 0.79 and 75 micrograms, respectively). In the hot water test, morphine induced analgesia with an ED50 value of 4.4 micrograms, whereas DPDPE failed to produce a full effect at doses up to 200 micrograms. Full dose-effect curves were also generated for various fixed-ratio combinations of DPDPE and morphine in both analgesic assays. Fixed ratios were chosen such that the amount of DPDPE in each dose of the combinations tested was itself subanalgesic. The combination with 20% DPDPE and 80% morphine (by weight) was significantly superadditive in the cold water test as determined by isobolographic analysis, whereas a second combination of the same drugs (40% DPDPE) was not. However, in the hot water test, the 20% DPDPE combination was not superadditive and neither were two other combinations of DPDPE and morphine. No combination of DPDPE and [N-MePhe3,D-Pro4]morphiceplin differed significantly from additivity when tested in the cold water test.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of dopamine antagonists on locomotor activity, conditioned activity and conditioned place preference induced by cocaine in rats.

Neuronal substrates that mediate the conditioned effects of cocaine have not been well characterized. To examine dopaminergic mechanisms, three antagonists were tested for their capacity to inhibit the expression of conditioned locomotor activity and conditioned place preference in rats. Antagonists were also assessed against acute cocaine-stimulated locomotor activity for comparison. For locomotor activity conditioning, six conditioning sessions were conducted over a 10-day period. Paired rats received 10 mg/kg cocaine prior to activity sessions and saline after; unpaired controls received saline prior and cocaine after. For place preference conditioning, eight conditioning sessions were conducted over a 13-day period; rats received 10 mg/kg cocaine while restricted to one of two distinct chambers and, on alternate days, they received saline in the other. Antagonists (haloperidol, raclopride and SCH23390; 0.03-0.1 mg/kg) were given only on test days for conditioned effects. All three antagonists significantly and dose-dependently attenuated the direct stimulatory effect of cocaine. SCH23390 showed a tendency to reduce the expression of conditioned locomotor activity, and only haloperidol blocked the expression of conditioned place preference. Thus, direct and conditioned stimulant effects of cocaine were shown to be differentially sensitive to dopamine receptor blockade. Further, conditioned stimulant effects differed from conditioned reinforcing effects in this regard.