Early neonatal experience of Long-Evans rats results in long-lasting changes in morphine tolerance and dependence.

RATIONALE: Daily, 3-h separations from the dam on postnatal days 2-14 produce long-lasting changes in behavioral and neuroendocrine responses to stressors and sensitivity to acute morphine in Long-Evans rats. OBJECTIVE: We tested whether offspring that were separated from their dam for 3 h daily (MS) on postnatal days 2-14 exhibit altered sensitivity to chronic morphine, compared to animals that experienced only brief (15 min) separations (H) from the dam or that were left undisturbed (NH) during the same period. METHODS: Subjects received 1 week SC infusion of either morphine, or saline via osmotic pumps. Twenty-hours after pump removal, the global opioid withdrawal scores were recorded. Four hours later, animals were tested for antinociception (tail-flick and hot-plate tests) during cumulative morphine administration. RESULTS: MS males and MS females undergoing withdrawal from chronic morphine had higher global withdrawal scores compared to NH controls. MS males (but not MS females) were less sensitive to the antinociceptive effects of morphine compared to H and NH controls, primarily in the hot-plate test, regardless of whether they had received a saline or a morphine infusion. MS males consistently exhibited significant morphine tolerance, whereas control males failed to exhibit tolerance either in the hot-plate test (NH group) or in both antinociception assays (H group). In contrast, tolerance was exhibited by all females in both tests for antinociception. CONCLUSIONS: These data indicate that repeated neonatal maternal separation alters sensitivity to chronic morphine administration in a sex-dependent manner.

Repeated neonatal maternal separation alters morphine-induced antinociception in male rats.

Recent studies indicate that Long-Evans rats separated from their dam for 3 h daily over the first 2 weeks of life (maternally separated [MS] rats) exhibit exaggerated behavioral and neuroendocrine responses to stress as adults compared to handled (H) or non-handled (NH) control animals. Our aim was to determine whether repeated neonatal maternal separation results in altered sensitivity to the opioid agonist morphine in male and female adult rats. Sensitivity to morphine was assessed using hot-plate and tail-flick tests. Morphine was less potent inducing antinociception in MS males compared to same-sex controls in the hot-plate, but not in the tail-flick test. Decrease in sensitivity to morphine in MS females compared to same-sex controls was present only as a trend in the hot-plate, but not in the tail-flick test. These results suggest that neonatal maternal separation results in long-lasting changes in opioid responsiveness primarily in male rats.

Central discriminative effects of morphine in rats: training via intracerebroventricular administration.

There have been studies of the discriminative effects of intracerebroventricularly (ICV)-administered morphine (MOR) in rats trained to discriminate MOR systemically, but the converse has not been done. In this study, rats were trained to discriminate between ICV (1-10 microg/3 microl, 1 h) or subcutaneous (SC) (3.0 mg/kg, 30 min) injections of MOR vs. saline/vehicle in a discrete-trial avoidance/escape procedure. On generalization testing, subjects in both the ICV- and SC-trained groups responded on the MOR-appropriate lever at ICV MOR doses < or =1-3 microg, and at SC MOR doses 2 to 3 orders of magnitude higher (vs. ICV). Naltrexone (SC) blocked the stimulus effects of MOR (ICV) equipotently in both training groups. In ICV-trained subjects, levorphanol (SC), the mu-opioid selective peptide [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAMGO) (ICV), and the enkephalinase inhibitor N-[L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (SCH 32615) (ICV) produced complete MOR-appropriate responding, whereas the dextrorotary enantiomer of levorphanol dextrorphan (SC; < or = 3.0 mg/kg) and the delta-opioid selective peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE) (ICV, < or = 0.03 mg) did not. SC-trained subjects did not generalize to SCH 32615, which suggests qualitative differences in the discriminative stimulus effects of novel drugs as a function of the route of administration of the training drug. These data demonstrate that it is feasible to train rats to discriminate an opioid administered by the ICV route, and to perform extended tests of generalization to novel drugs (SC or ICV) in rats so trained.

Periodic postpartum separation from the offspring results in long-lasting changes in anxiety-related behaviors and sensitivity to morphine in Long-Evans mother rats.

RATIONALE: According to recent studies daily, 3-h separations from the dam on postnatal days 2-14 produce long-lasting changes in responses to stressors and sensitivity to morphine in Long-Evans offspring. It has not been investigated whether daily dam-litter separations can have long-lasting effects also on the mother beyond weaning of the pups. OBJECTIVE: We tested whether dams that were separated from their litter for 3 h daily (LS) on postpartum days 2-14, like their offspring, exhibit altered anxiety-like behaviors and sensitivity to morphine, compared to dams that experienced only brief (15-min) separations (BS) from the litter or that were left undisturbed (NH) during the same period. METHODS: Four to six weeks after weaning, subjects were tested on the elevated plus-maze, in a novel locomotor activity arena, or were exposed to loud auditory stimuli. Sensitivity to morphine was assessed using hot-plate and tail-flick tests. RESULTS: LS dams spent significantly more time in the open arms of the plus-maze and in the center of the locomotor activity arena, and were more likely to emit ultrasonic vocalizations in response to auditory startle stimuli compared to NH dams. Furthermore, LS dams were less sensitive to morphine, primarily in the tail-flick test. Dams that experienced brief litter separations (BS), like LS dams, exhibited altered performance on the plus-maze and elevated ultrasonic vocalizations. However, BS dams were similar to NH controls in locomotor activity and sensitivity to morphine. CONCLUSIONS: Multiple postpartum separations from the offspring alter the behavior of Long-Evans dams in novel/aversive environments and affect their sensitivity to the antinociceptive effects of morphine.

Acute opioid but not benzodiazepine dependence in rats responding for intracranial self-stimulation.

RATIONALE: Four-hour pretreatment with a single dose of morphine or related opioids sensitizes rats responding for intracranial self-stimulation (ICSS) to the rate-decreasing effect of naltrexone, indicative of antagonist-precipitated withdrawal from acute opioid dependence. OBJECTIVES: To determine whether sensitization to naltrexone could be observed in morphine-pretreated rats responding under a progressive ratio (PR) schedule of ICSS and to determine whether acute pretreatment with benzodiazepines produces similar sensitization to flumazenil. METHODS: Rats with an electrode in the medial forebrain bundle were trained to respond under an ICSS PR schedule, in which the number of responses required for a 250-ms stimulus started at one, then increased gradually. If no responding occurred for 30 s, the response requirement reverted to a single response and the break point was operationally defined. RESULTS: Pretreatment (4-h) with 3.0 mg/kg or 5.6 mg/kg morphine reduced the ED25 values of naltrexone for decreasing response rate from 18+/-6.7 mg/kg to 0.021+/-0.006 mg/kg and 0.006+/-0.001 mg/kg, respectively. Changes in break point usually paralleled changes in response rate. In contrast, 4- to 24-h pretreatment with the benzodiazepines chlordiazepoxide (30 mg/kg and 100 mg/kg) or diazepam (3.0 mg/kg and 10 mg/kg), behaviorally-active doses, did not significantly alter sensitivity to the effects of flumazenil (1.0-30 mg/kg). CONCLUSIONS: These results show that PR ICSS provides a stable behavioral baseline for testing drugs in rats and extend to this procedure the generality of the phenomenon of acute opioid dependence. There was no comparable evidence of acute benzodiazepine dependence, suggesting that there are differences in the ways that opioid and benzodiazepine agonists initiate the adaptive changes that underlie the state of physical dependence.

Sensitization to daily morphine injections in rats with unilateral lesions of the substantia nigra.

Morphine indirectly enhances dopaminergic activity in the nigrostriatal system, and repeated administration of morphine progressively increases the locomotor activity of rats. We used the rotational behavior model to determine if daily morphine produces an increase in turning and produces cross-sensitization to d-amphetamine and cocaine. Rats with unilateral nigrostriatal lesions received daily injections of saline or morphine (10 mg/kg). Repeated morphine administration produced a progressive increase in turning over 13 days. Next, a morphine dose-response curve (1.0-30 mg/kg) was determined. Both the saline and morphine-treated groups showed dose-dependent increases in turning, but, the peak effect in the morphine group was higher than that in the saline group, indicating sensitization to morphine. The morphine-treated group did not show cross-sensitization to either d-amphetamine (0.1-3 mg/kg) or cocaine (1.0-30 mg/kg); in fact, it showed less cocaine-induced turning than the saline group. Seventy-one days after saline or morphine injections began, the morphine group was still significantly more sensitive to turning induced by 10 mg/kg morphine than the saline group was (200 vs. 750). Therefore, repeated daily injections of morphine produce a progressive sensitization to turning induced by morphine in the absence of cross-sensitization to turning induced by psychomotor stimulants.

Discriminative stimulus effects of naltrexone after a single dose of morphine in the rat.

The discriminative stimulus effects of an acute morphine (MOR) --> naltrexone (NTX) combination were characterized and compared with the stimulus effects of NTX-precipitated and spontaneous withdrawal from chronic MOR administration. Adult male Sprague-Dawley rats (n = 6-8) were trained to discriminate between two drug treatments in a discrete-trial avoidance/escape procedure: MOR (10 mg./kg, s.c., 4 h) --> NTX (0.3 mg/kg, s.c., 0.25 h) versus saline (SAL, 1 ml/kg, s. c., 4 h) --> NTX (0.3 mg/kg, s.c., 0.25 h). Subjects responded only on the SAL --> NTX-appropriate lever when SAL was given 3.75 h after MOR or 3.75 h before any dose of NTX (0.3-100 mg/kg). Responding was dose dependent and MOR --> NTX-appropriate when NTX (0.01-0.1 mg/kg) followed MOR. Full MOR --> NTX-appropriate responding was dependent on the pretreatment dose and time of MOR, with full effects observed only when MOR (10 mg/kg) was given 3 to 4 h before NTX. While subjects were maintained on either 20- or 40 mg/kg/day of MOR via osmotic pump, NTX produced full dose-dependent, MOR --> NTX-appropriate responding. When the MOR-filled pumps were removed, partial MOR --> NTX-appropriate responding occurred, peaking at 6 to 12 h. The physical withdrawal signs produced by NTX after acute or during chronic MOR exposure were of smaller magnitude compared with the ones that occurred during abrupt withdrawal from chronic MOR. A qualitatively unique "withdrawal" stimulus that is dose- and time-dependent appears to be the basis of this MOR --> NTX discrimination.

Comparison of the discriminative and antinociceptive effects of morphine and its glucuronide metabolites after central or systemic administration in the rat.

The potential of centrally (ICV) or systemically (SC) administered M6G to substitute for morphine in a drug discrimination task was characterized in the present study. Rats with a cannula in the lateral cerebral ventricle were trained to discriminate between injections of morphine (3 mg/kg, SC) and saline using a discrete-trial avoidance/escape procedure. Substitution tests were conducted with SC or ICV morphine, morphine-3-beta-D-glucuronide (M3G), or morphine-6-beta-D-glucuronide (M6G) and response latency in a tail-flick test was measured before each session began. The stimulus effects of morphine (ED50=1.02 mg/kg SC or 2.1 microg/kg ICV) were fully shared by M6G, with potency dependent on route of administration (ED50=3.12 mg/kg SC or 0.34 microg/kg ICV). The stimulus effects of M6G were highly correlated with its antinociceptive activity (r=0.84 SC or 0.46 ICV) and, at equipotent systemic doses, they lasted longer (t1/2=391 min) than those of morphine (t1/2=185 min). M3G was inactive in both procedures by both routes of administration. Naltrexone SC, given 30 min prior to testing, completely attenuated the stimulus effects of ICV M6G (AD50=0.011 mg/kg), indicating that they are mediated by opioid receptors. The results of this study suggest that M6G might contribute to the interoceptive effects of morphine that underlie its potential for abuse.

Intracranial self-stimulation in rats: sensitization to an opioid antagonist following acute or chronic treatment with mu opioid agonists.

Acute mu opioid agonist pretreatment (4 hr) dose-dependently sensitizes rats responding for food reinforcement to the rate-decreasing effects of naltrexone (NTX). In the present study, adult rats were trained to respond in an intracranial self-stimulation autotitration procedure in which responding resulted in electrical stimulation of the medial forebrain bundle that decreased in frequency until reset to the initial value. In an acute sensitization experiment, pretreatment (4 hr) doses of 3.0 and 10 mg/kg morphine reduced the ED25 value for the intracranial self-stimulation rate-decreasing effect of NTX from 28.2 mg/kg to 0.29 and 0.02 mg/kg, respectively. All mu-selective opioid agonists tested, fentanyl > levorphanol > methadone > morphine > meperidine (listed in order of decreasing potency), produced similar large increases in sensitivity to NTX. Acute sensitization was not induced by the kappa-selective opioid agonist spiradoline, the dextrorotary enantiomer of levorphanol, dextrorphan, or the nonopioid drugs d-amphetamine and pentobarbital. Pretreatment with morphine for 10 days by continuous subcutaneous infusion (15 mg/kg/day) reduced the ED25 value of NTX from 28.2 to 0.002 +/- 1.48 mg/kg. The correlation of decreases in ED25 values for the rate-decreasing effect of NTX after both acute and chronic morphine administration is consistent with the theory that acute agonist-induced sensitization reflects receptor-mediated changes occurring early in the development of physical dependence.

Parametric changes in response equilibrium during an intra-cranial self stimulation (ICSS) task: can reward value be assessed independently of absolute threshold?

Traditional ICSS methodologies have attempted to evaluate changes in the rewarding value of brain stimulation by assessing the lowest value of the stimulation that will support responding. However, orderly changes in suprathreshold indicants of hedonic magnitude such as titration point have been shown. In the present experiments, rats were trained to respond on two ICSS autotitration schedules in which every response on one lever produced stimulation of the medial forebrain bundle, and every Xth response decreased either the stimulation current or the stimulation frequency. At any time, a response on a second "reset" lever restored the stimulation current or frequency available on the stimulation lever to its starting level and operationally defined changes in "reward value". In order to study this titration point measure, two response requirements (responses/stepdown; step size) and two stimulation parameters (initial stimulation level; train duration) were systematically varied. Under both current and frequency titration schedules, data indicated that response rate and titration point remained stable over repeated trials and multiple testing days--parameters being constant. Across all conditions, compared to the frequency titration schedule, subjects responding under the current titration schedule showed significantly higher titration points and lower rates of responding. Indicating the independence of rate and titration point data, parametric manipulations did not affect titration point and rate data concurrently. Results support the conclusion that titration point is a relative measure of "reward value" that is generally independent of response rate, but that is affected by manipulations that alter the amount of stimulation available between "resets". Additional work is needed in order to determine the relationship between the magnitude of stimulation needed to maintain minimal responding and that needed to maintain response equilibrium in an autotitration task.