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Chronic cadmium exposure attenuates the conditioned reinforcing properties of morphine and fentanyl
Brain Research 776 Ž1997. 162–169
Research report
Chronic cadmium exposure attenuates the conditioned reinforcing properties of morphine and fentanyl Dennis K. Miller, Jack R. Nation
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Department of Psychology, Texas A & M UniÕersity, College Station, TX 77643, USA Accepted 12 August 1997
Abstract Adult male rats were exposed ad libitum for 40 days to 100 ppm cadmium chloride through their diet, or an identical diet with no added cadmium. Conditioned place preference ŽCPP. was conducted in a 2-chamber apparatus in which all drugs were paired with the least-preferred side as determined on a pre-test. In Experiment 1, control and cadmium-exposed rats received 0, 0.6, 1.25, 2.5, or 5 mgrkg morphine sulfate Ži.p.. for 4 days, and vehicle only for 4 days. Control animals showed a preference for the drug-paired side at 1.25, 2.5, and 5 mgrkg while the cadmium-exposed rats showed a preference at 5 mgrkg only. In Experiment 2, rats were implanted with cannulae into the lateral ventricles and 0, 2, or 5 mg morphine sulfate was administered intracerebroventricularly Ži.c.v... An attenuation by cadmium again was observed, as control animals showed a place preference at 2 and 5 mg and cadmium-exposed animals showed preference at 5 mg only. In Experiment 3, increasing doses of the m-opioid receptor agonist fentanyl Ž0, 0.0004, 0.004, and 0.04 mgrkg. were systemically administered Žs.c.. and rats tested for CPP. While cadmium animals showed place preference only at 0.04 mgrkg, control animals showed preference at 0.0004, 0.004, and 0.04 mgrkg. These findings are discussed within the framework of metal-induced disturbance of neurochemical function andror associative processing, and the implications that such disturbances may have for drug seeking and taking. q 1997 Elsevier Science B.V. Keywords: Behavior; Cadmium; Fentanyl; Morphine; Opiate; Place preference
1. Introduction Cadmium is a widely distributed metal that selectively accrues in tobacco leaves w30x. Not surprisingly, cigarette smoking has been shown to elevate the body burden of cadmium to a level twice that of non-smokers w22,29,30x. The effects of long-term exposure to cadmium are significant as the metal is efficiently retained in the body and accumulates with age w10x. Due to the higher body burdens of cadmium in smokers, and a correlation between tobacco use and other drug taking w24x, it is important to examine the potential interactions of this heavy metal with drugs of abuse. Using an animal model, chronic cadmium exposure, at blood tissue levels that approximate smokers, has been shown to increase the intake of ethanol w18x and cocaine Abbreviations: CPP, conditioned place preference; DA, dopamine; NA, nucleus accumbens; VTA, ventral tegmental area; NMDA, Nmethyl-D-aspartate ) Corresponding author. Fax: q1 Ž409. 845-4727; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 1 3 - 5
w16x. Additionally, it has been reported recently that animals exposed to chronic cadmium show an attenuation to the locomotor decreasing effects of acute systemic morphine administration, as well as the locomotor increasing Žbehavioral sensitization. effects of morphine associated with repeated drug exposures w17x. The behavioral sensitization effects produced by repeated opioid administration appear to be mediated by an increase in activation of dopamine ŽDA. projections in the nucleus accumbens ŽNA.. This increase may derive from N-methyl-D-aspartate ŽNMDA. receptor modulation of DA pathways, andror activation of ventral tegmental area ŽVTA. m-opioid receptors that compromise the inhibitory influence of GABA interneurons, ultimately causing an increase in DA activity w9x. The established attenuation of morphine locomotor sensitization by cadmium could be due to an antagonism at any part of this proposed pathway. Cadmium blocks voltage-dependent NMDA channels w12x, decreases the stimulated release of DA w20x, and has been demonstrated to act as a competitive m-opioid antagonist w27x. It has been difficult to separate the effects of the metal on these systems using behavioral techniques. Few
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behavioral studies of cadmium toxicity have involved local applications of the metal, and given that toxicant clearance cannot be accomplished effectively in the intact animal w10x, it is unlikely that such studies would yield useful information. Regardless of the neural loci associated with cadmiumropiate interactions, the pattern of results produced by cadmium exposure has profound implications with respect to defining the abuse potential of morphine and other addictive drugs that operate via activation of opioid receptor subtypes. Along these lines, considerable research exists to demonstrate the importance of drug-related stimuli in controlling the behavior and subjective perceptions of drugs in humans, and an appropriate paradigm to study such motivational factors underlying drug use is conditioned place preference ŽCPP.. In this model, a drug is administered to the animal immediately before placement in an environment with unique contextual stimuli Žolfactory, visual, tactile.. Following several pairings of the drug and the unique context, and separate pairings of a distinctively different context and no-drug Žvehicle., the animal is tested for preference by being allowed free-choice access to the drug-paired and the vehicle-paired context. CPP is then defined by some measure of preference for one environment over another. In this regard, morphine and other opioid or non-opioid compounds have been shown to produce a robust CPP in animals that have not been exposed to exogenous chemical contamination w8x. The m-opioid receptor is believed to be influential in the reinforcing properties of opiates, and as indicated, numerous reports are available which show systemic administration of morphine w1x or the selective m-opioid agonist fentanyl w14x produce place preference. Inasmuch as cadmium acts as a m-receptor antagonist, it follows that cadmium exposure should diminish the reinforcing properties of m-opioid agonists such as morphine and fentanyl, and therein alter performance on a CPP task. In addition to the essential role of the m-receptor subtype in the development of CPP, it is established that disturbances in DA function following NA lesions by 6-OHDA w25x have parallel effects in terms of attenuating the rewarding effects of opiates. The NMDA antagonist MK-801 has also been shown to attenuate CPP, although its impact on morphine reinforcement versus associative learning has yet to be determined w28x. Because of the aforementioned disrupting effects of cadmium on NMDA and DA systems w12,20x, there is increased justification for pursuing issues relating to cadmium-related changes in the reinforcement efficacy of opioid agonists. Accordingly, employing a CPP preparation, the present report presents findings from a series of investigations on the effects of chronic dietary cadmium exposure on the conditioned reinforcing effects of the m-opioid receptor agonists morphine and fentanyl. Experiments 1 and 2 assessed the effects of chronic cadmium exposure on
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systemic Ži.p.. and intracerebroventricular Ži.c.v.. administered morphine on CPP, and Experiment 3 examined the effects on systemic Žs.c.. fentanyl on CPP.
2. Materials and methods 2.1. Apparatus Place conditioning and testing was conducted in seven 20 = 60 = 20 cm wooden shuttle boxes with wooden tilt floors. At each end of a box was a microswitch interfaced with an IBM compatible computer. A BASIC computer program was written to continuously record the number of times and duration the switch was activated through a tilt of the floor by a rat. Half the box had white walls with a smooth white floor and the other had black walls with a black sandpaper floor. For conditioning sessions, the boxes were divided into two equal-sized compartments by removable partitions. On test sessions the partitions were removed and a 20 = 10 = 5 cm wooden platform was installed 2 cm above the floor to divide the two compartments but allow free access by rats. Subjects showed a strong preference for the black side in experiments conducted before those presented here. To attenuate this preference, a 40 W light was positioned 50 cm above the black side of each apparatus. These seven lamps provided the only illumination in the room. Following each conditioning and test session the apparatus was cleaned with a mild soap solution. The apparatus was located in a sound-resistant room with a 40 dB white noise generator operating continuously. 2.2. Drugs Morphine sulfate was provided gratis by the Research Technology Branch of NIDA. For i.p. administration, the drug was dissolved in a filtered water vehicle, and the dosage is expressed as the salt. The morphine sulfate for i.c.v. administration was dissolved in a Ringer’s solution made of 120 mM NaCl, 2 mM CaCl 2 , and 2 mM NaHCO 3 in sterile water. Fentanyl ŽSigma Co.; St. Louis, MO, USA. was dissolved in a 0.9% saline vehicle and the dosage is expressed as the base from fentanyl citrate. The drug was administered s.c. 2.3. Experiment 1: systemic morphine administration 2.3.1. Subjects The subjects used in this study were 93 male SpragueDawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival at the laboratory. Body weights ranged from 175 to 199 g. Forty-six animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 47 animals received ad libitum access to an identical diet, except that it con-
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tained 100 ppm cadmium chloride. Both were specially prepared by Dyets, Inc. ŽBethlehem, PA, USA.. Continuous access to tap water was available in the home cages. Throughout the experiment, animals were double-housed in hanging polycarbonate cages. Each cage contained two members of the same exposure group Žcadmium or control diet.. The cages were located in a temperature and humidity controlled animal colony with continuous light exposure. Food consumption and body weights were recorded weekly throughout the experiment. Individual animal food consumption was measured by halving the total consumed by the cage pair. Behavioral training and testing sessions were conducted at 08:00 h. The animal holding and testing facility is approved by the Association for Assessment and Accreditation of Laboratory Animals Care International ŽAAALAC International., and all animal maintenance and research was conducted in accordance with the guidelines provided by the University Laboratory Animal Care Committee ŽULACC.. The health of the animals was monitored throughout the duration of the project by the campus veterinarian.
2.3.2. Procedures for Experiment 1 Following 40 days of pre-exposure to their appropriate control or cadmium diets, the animals began CPP conditioning and testing. On Day 1 of the study, animals were transferred from the colony to the testing room for 40 min to habituate to the sound and illumination of the room. They were not placed in the CPP apparatus. Initial biases for the white or black chamber Žpre-test. were determined on Day 2 as non-injected animals were given free access to either chamber for 15 min. On Days 3–10 the animals received one of five injections of morphine Ž0, 0.6, 1.25, 2.5, or 5 mgrkg, i.p.. on four of the conditioning days, and four vehicle Žwater. injections on the remaining four conditioning days. All injections were given at a volume of 1 mlrkg. The animal was confined to the side least preferred Ždefined as the side in which the animal spent the least amount of time on the Day 2 pre-test., 5 min after receiving a morphine injection. The animal was confined to the most-preferred chamber Ždefined as the side in which the animal spent the most amount of time on the Day 2 pre-test., 5 min following a vehicle injection. Drug and vehicle were presented on alternating days and the injection received first was counterbalanced for each dose and exposure regimen Žcadmium or control.. Animals were run in squads of seven, counterbalancing by dose and group assignment. The period of confinement for each conditioning trial was 40 min. A post-test was conducted on Day 11 following the same procedure as the Day 2 pre-test. Twenty-four hours after the post-test, animals were rendered unconscious with 60 mgrkg sodium pentobarbital Ži.p.., decapitated, and approximately 3–5 ml of trunk blood was collected.
2.4. Experiment 2: intracerebroÕentricular (i.c.Õ.) morphine administration 2.4.1. Subjects The subjects used in Experiment 2 were 45 male Sprague-Dawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival at the laboratory. Body weights ranged from 175 to 199 g. Twenty-three animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 22 animals received ad libitum access to an identical diet, except that it contained 100 ppm cadmium chloride. All other aspects of animal maintenance were as described in Experiment 1. 2.4.2. Procedures for Experiment 2 Following 40 days of pre-exposure to their appropriate cadmium or control diets, each animal was implanted stereotaxically with a unilateral guide cannula at the following stereotaxic coordinates: 1.0 mm posterior and 4.2 mm ventral from Bregma, and y1.8 mm from the midline w21x. The guide cannula was 13 mm of 22-gauge stainless steel tubing protected by a 30-gauge stainless steel obturator. Anesthesia was produced by 50 mgrkg Ži.p.. sodium pentobarbital. Following surgery, animals were housed singly through the completion of the experiment. Conditioning and testing began 1 week after surgery. Procedures for Days 1 and 2 were identical to the previous experiment. On Days 3–6 each obturator was removed and a 30-gauge injector Ž18 mm. was inserted through the outer guide cannula. After 1 min each animal was infused with 0 ŽRinger’s solution., 2, or 5 mg morphine in a volume of 1 ml. All solutions were administered by hand over a 15 s period using a microliter syringe. Immediately after receiving a drug injection the animal was confined to the side least preferred as determined on Day 2. Immediately after receiving a vehicle injection the animal was confined to the most-preferred chamber. The period of confinement was 50 min. A post-test was conducted on Day 7 following the same procedure as the Day 2 pre-test. Twenty-four hours following testing, each animal was sacrificed with pentobarbital. Approximately 3–5 ml blood was collected via a cardiac puncture and then the animal was immediately perfused with 10% formalin. Before removing the brain, 2 ml of cresyl violet ŽSigma Co.; St. Louis, MO, USA. in distilled water solution was injected through the cannula. After 10 min, the brain was removed and a transverse slice was made 1 mm posterior of the cannula site. The third ventricle was visually examined for the presence of the colored solution as an initial verification of appropriate cannulae placement. Brains were prepared in a formalin solution and 1 week later, cannula tracts were confirmed by histological preparation of 50 mm transverse slices. The behavioral data collected for each animal were used in the analysis only if the cresyl
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violet was present and the histology indicated the cannula was in the lateral ventricle. 2.5. Experiment 3: systemic fentanyl administration 2.5.1. Subjects The subjects used in Experiment 3 were 52 male Sprague-Dawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival to the laboratory. Body weights ranged from 175 to 199 g. Twenty-six animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 26 animals received ad libitum access to an identical diet, except that it contained 100 ppm cadmium chloride. All other aspects of animal maintenance were as described in Experiment 1. 2.5.2. Procedures for Experiment 3 The conditioning and testing procedures for Experiment 3 were precisely as described for Experiment 1, with the exception that animals received one of four fentanyl doses Ž0, 0.0004, 0.004, and 0.04 mgrkg, s.c.. on the drug-side of the chamber during the training period ŽDays 3–10.. The fentanyl injections were given 10 min prior to placement in the chamber and the duration of confinement was 20 min. All other aspects of the study, including post-test procedures on Day 11, were as described for Experiment 1.
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sis of variance ŽANOVA. test was employed, and simple effects analyses and Newman Keuls post hoc tests were used when appropriate to determine significant differences Ž P - 0.05..
3. Results 3.1. Body weights and food consumption For each of the three experiments, there were no significant differences in body weights or food consumption according to exposure group Žcadmium or control. or dose of drug administered, as determined by ANOVAs of Group = Week and Dose = Group Ž P ) 0.05.. Body weights did increase systematically by week for all animals, and at the start of behavioral testing, body weights ranged from 440 to 530 g. Average cadmium exposure was approximately 10.0 mgrkgrday for animals receiving the adulterated diet. 3.2. Experiment 1: systemic morphine administration The animals in both exposure groups spent more time in the black chamber on the Day 2 pre-test. There were no significant differences Ž t 87 s 0.72, P ) 0.05. between cadmium-exposed Žmean s 231.1 s, S.E.M.s "22.3 s. and control Žmean s 254.4 s, S.E.M.s "23.2 s. animals with
2.6. Cadmium residues in blood Following collection of blood samples, cadmium residues in blood were measured via atomic absorption spectrophotometry w15x. 2.7. Data analysis Conditioning scores were defined by the change in time Žmeasured in s. in the drug-paired chamber from the pre-test to post-test Žtime on post-test minus time on pre-test.. A conditioned place preference occurred when the mean group conditioning score was significantly greater than the vehicle-only Ž0 mgrkg or 0 mg. group. Comparisons were made within exposure groups Žcontrol or cadmium.. For example, cadmium-morphine dose groups were compared to the cadmium vehicle-only group. Additional comparisons of group differences were made at each dose in an effort to more explicitly detail points of separation between control and cadmium-exposed animals. Four animals in Experiment 1, 7 animals in Experiment 2, and 2 animals in Experiment 3 showed a strong preference Ždefined as greater than 800 s in the 900 s pre-test. for one side over another on the Day 2 pre-test. These animals were not included in any data analysis. In addition, 2 animals in Experiment 2 were excluded due to incorrect cannulae placements. For each experiment, a Groups= Dose factorial analy-
Fig. 1. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for morphine Ži.p.. CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
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respect to the amount of time spent in the side least-preferred on the pre-test. A two-way ANOVA ŽExposure Group = Morphine Dose. was conducted on the conditioning scores yielded by Day 11 testing Žsee Fig. 1.. A significant main effect of Morphine Dose was found Ž F4,79 s 7.15, P - 0.001. and post hoc tests indicated the 5 mgrkg dose Žmean s 323.6 s, S.E.M.s "44.6 s. had greater conditioning scores than the vehicle-only Žmean s 114.2 s, S.E.M.s "32.4 s. dose. A significant main effect of Exposure Group was also found Ž F1,79 s 10.87, P - 0.01. as the control diet exposure group Žmean s 276.1 s, S.E.M.s "24.9 s. showed greater overall conditioning scores than the cadmium diet group Žmean s 182.6 s, S.E.M.s "21.3 s.. Based on a simple main effect analysis and the appropriate post hoc tests, the control animals showed significant place preference at 1.25, 2.5, and 5 mgrkg Ž F4,40 s 6.74, P - 0.05.. Within the cadmium exposure group, a significant place preference was found only at the 5 mgrkg dose as indicated by the simple main effect of dose Ž F4,39 s 2.61, P - 0.05. and post hoc tests Žsee Fig. 1.. Additional comparisons of group separation at each dose of morphine found that the control diet exposure group showed significantly greater conditioning scores than the cadmium exposure group at the 1.25 Ž t 16 s 2.82, P - 0.05. and 2.5 mgrkg Ž t 20 s 3.83, P - 0.01. doses. A marginally significant difference was seen at the 0.6 mgrkg Ž t 12 s 2.12, P s 0.055. dose. These differences in dose required to produce place preference between groups indicate a shift to the right in the dose effect curve for morphine CPP in cadmium-exposed animals. 3.3. Cadmium residues in blood For Experiment 1, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 9.16 mgrdl, S.E.M.s "3.68 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 87 s 20.01, P - 0.001.. 3.4. Experiment 2: intracerebroÕentricular (i.c.Õ.) morphine administration As in Experiment 1, both groups Žcontrol mean s 273.3 s, S.E.M.s "31.0 s; cadmium mean s 273.2 s, S.E.M.s "30.9 s. had an overall preference for the black chamber on the Day 2 pre-test. There were no differences between groups on the amount of time spent in the side least-preferred on the pre-test Ž t 34 s 0.01, P ) 0.05.. A two-way ŽExposure Group = Morphine Dose. ANOVA was conducted on the conditioning scores Žsee Fig. 2., and as in the previous experiments a significant main effect of Exposure Group was found Ž F1,30 s 8.29, P - 0.01.. Control animals Žmean s 202.9 s, S.E.M.s "45.3 s. had significantly greater conditioning scores than
Fig. 2. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for morphine Ži.c.v.. CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
cadmium-exposed animals Žmean s 47.23 s, S.E.M.s "42.3 s.. A significant main effect of Morphine Dose was also found Ž F2,30 s 3.82, P - 0.05.. Greater conditioning scores were shown at the 5 mg dose Žmean s 238.2 s, S.E.M.s "45.8 s. than vehicle-only Žmean s 48.8 s, S.E.M.s "54.5 s.. Based on a simple effects analysis and the appropriate post hoc tests a significant place preference Ž F2,17 s 4.71, P - 0.05. was shown at 2 and 5 mg for control animals. Cadmium-exposed animals also showed a significant effect of Morphine Dose Ž F2,15 s 6.78, P - 0.01. but only the 5 mg dose was different from vehicle-only. Additional comparisons of group differences at each dose found that control animals showed significantly greater conditioning scores at the 2 mg dose than cadmium-exposed animals Ž t 34 s 4.76, P - 0.01.. As with Experiment 1, a shift to the right in the dose-effect curve was seen in cadmium-exposed animals. 3.5. Cadmium residues in blood For Experiment 2, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 8.86 mgrdl, S.E.M.s "4.35 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 34 s 18.40, P - 0.001..
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3.6. Experiment 3: systemic fentanyl administration As with Experiments 1 and 2, both groups had an overall preference for the black chamber on the Day 2 pre-test. Again, there were no differences between groups Žcontrol mean s 328.6 s, S.E.M.s "16.3 s; cadmium mean s 308.9 s, S.E.M.s "23.0 s. on the amount of time spent in the side least-preferred on the pre-test Ž t 48 s 0.67, P ) 0.05.. A two-way ŽExposure Group= Fentanyl Dose. ANOVA was conducted on the fentanyl conditioning scores ŽFig. 3.. A significant main effect of Exposure Group was found Ž F1,42 s 6.75, P - 0.05., as control animals Žmean s 226.2 s, S.E.M.s "31.7 s. had greater overall conditioning scores than cadmium-exposed animals Žmean s 135.8 s, S.E.M.s "27.8 s.. A significant main effect of Fentanyl Dose was also found Ž F3,42 s 8.38, P - 0.001.. Greater conditioning scores were shown at the 0.04 mgrkg concentration Žmean s 296.6 s, S.E.M.s "27.6 s. than vehicle only Žmean s 53.7 s, S.E.M.s "26.0 s.. Based on the simple effects analysis and post hoc tests control animals showed a significant place preference at the 0.0004, 0.004, and 0.04 mgrkg dose Ž F3,22 s 4.56, P - 0.05.. A significant effect of dose also was found with cadmium-exposed animals Ž F3,20 s 5.68, P - 0.01., but only the conditioning score for the 0.04 mgrkg dose of
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fentanyl was different from the vehicle-only group. Additional comparisons of groups at each dose found that control animals showed significantly greater conditioning scores at the 0.004 mgrkg dose than cadmium-exposed animals Ž t 11 s 3.25, P - 0.01.. Thus, as with the nonselective m-opioid agonist morphine, the dose-effect curve for fentanyl was shifted to the right with cadmium exposure. 3.7. Cadmium residues in blood For Experiment 3, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 8.30 mgrdl, S.E.M.s "3.68 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 48 s 20.02, P - 0.001..
4. Discussion An attenuation of the conditioned reinforcing properties of systemically administered morphine and fentanyl was observed for adult male rats exposed to chronic dietary cadmium. A complete blockade of CPP responding was not evident, however, as the cadmium-exposed animals did show a place preference, but at higher doses than controls. Additional evidence revealed that this pattern of attenuation is likely due to an antagonism by cadmium in the central nervous system, rather than a peripheral disruption of absorptionrdistribution of the drug, as a similar behavioral shift to the right in the dosereffect function was seen with morphine injected into the periphery and directly into the brain. 4.1. Systemic compared to i.c.Õ. morphine administration
Fig. 3. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for fentanyl CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
The parallel findings of Experiments 1 and 2 are instructive with respect to issues relating to the locus of cadmiumropiate interactions. Because kidney and liver are target organs of cadmium toxicity w6x, physiologic dysfunction stemming from enzymatic change or other disturbances in processes linked to pharmacodynamics may have reduced the availability of the morphine at neural sites, and therein altered the impact of a given amount of the drug. Insofar as peripheral changes limited CNS penetration, the attenuation effects observed here would be defined more appropriately as metal-based changes in drug presence rather than drug potency. Because it was demonstrated in Experiment 2 that place preference resulting from i.c.v. administration of morphine was attenuated by cadmium exposure, a drug availability account of cadmium-induced attenuation of the effects of morphine is rendered less likely, at least as a sole determinant of the interaction. An alternative interpretation rests with the demonstrated effects of cadmium on neural mechanisms that play a
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modulatory role in the development and expression of the behavioral effects of morphine. In this regard, the established antagonistic actions of cadmium on DA release w20x, excitatory amino acid activation of NMDA receptors w12x, and m-opioid sites w27x gain in significance with respect to interpretive issues. Disruption of any or all of these mechanisms w2,25,28x surely could produce the patterns of attenuation observed here and elsewhere w17x. In the present case, given that cadmium did attenuate CPP following centrally administered morphine, it seems reasonable to argue that toxicant-based disturbances in transmitter activity, binding affinity, etc., are integral to the pattern of behavioral antagonism of morphine CPP. This is not to say, of course, that metal-induced changes in metabolic processes or other non-CNS events are necessarily unrelated to cadmiumropiate interactions. Rather, the findings from Experiment 2 underscore the fact that the toxic effects of the contaminant on CNS operations must be considered as a prominent component of the overall influence of cadmium on morphine sensitivity. 4.2. Fentanyl administration Related to the matter of elucidating the mechanism of action of cadmium in the brain, the data from Experiment 3 may be of value. Although it is understood that morphine preferentially acts on the m-receptor, it also is recognized that other receptor types such as the d-opioid are affected by the drug w23x, and can be influential in the development of morphine-induced CPP w26x. The findings from Experiment 3 which show that CPP produced by systemic administration of the selective m-opioid agonist fentanyl is attenuated by cadmium point to this receptor site as a critical interface in the action of the metal in the CNS. Along these lines it would be of interest to know if a selective m-agonist such as DAMGO when applied directly to a region rich in m-receptors, such as the VTA, might reverse some of the behavioral effects associated with cadmium exposure. 4.3. InterpretiÕe issues It must be acknowledged that when a biased procedure is employed, as was the case here, there are potential difficulties associated with interpreting the data. That is, an increase in time spent on the drug-paired side of the apparatus may derive either from the conditioned reward effects of the drugs, or a diminished sensitivity to the aversiveness of the least-preferred place. Whatever the underlying reasons for the change in approach behavior, in the present experiments it is clear that cadmium has a profound impact on motivational stimuli associated with morphine and fentanyl. Also, it should be recognized that the effects observed in each of the three experiments reported here may derive from challenges to associative or cognitive processing rather than biologic mechanisms. CPP is a learned phe-
nomena in which the contextual cues of the environment acquire secondary reinforcing properties via classical conditioning w3x. It has been shown that blockade of glutamate neurotransmission by MK-801 impairs attention to exteroceptive stimuli w5x, and that compromises of NMDA activity by MK-801 retard behavioral plasticity and limit associative formations in a learning context w4x. Because cadmium acts as an NMDA antagonist w12x it is reasonable to assert that the effects of the metal on opiate-induced CPP were due to alterations in learning and conditioning mechanisms rather than drug action. Still, given the fact that similar effects have been observed in behavioral preparations Žlocomotor activity. where non-associative processes dictate the response profile w17x more pharmacologic accounts of our present findings likely are preferred. 4.4. Implications Finally, it is important to establish the relevancy of the present findings for the population of humans who abuse opiates. The cadmium residue levels in blood of the metal-exposed animals in this study approximate those of pack-a-day smokers w13x. Because the overall pattern of attenuation in this report on opiate CPP and a prior motor activity study w17x are indicative of opiate subsensitivity in toxicant-exposed rats, risk factors associated with drug selection and self-administration must be addressed. Numerous investigations of drug self-administration have shown that antagonism of high doses of opiates results in increased responding w7,19x, and for opiate self-administration this translates into a health threat. The elevated drug amounts produced by behavioral antagonism may reach toxic levels even though overt symptomotology is masked. The situation is likely worse when cadmium is the antagonist, inasmuch as cadmium has been shown to alter hepatic morphine-3-glucuronide ŽM3G. production w11x and thus disturb morphine metabolism. For drug abusers using tobacco products, and this is the bulk of the sub-population w24x, this could mean an increase in the pathologic consequences of drug taking. The increased cadmium burden associated with tobacco use w22,29,30x and attendant rise in opiate self-administration, coupled with an exacerbated toxicity profile, may define a status of enhanced vulnerability to the negative effects of these psychoactive drugs.
Acknowledgements This research was supported by United States Public Health Service Grant DA07932.
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D.K. Miller, J.R. Nationr Brain Research 776 (1997) 162–169 w2x M.D. Britt, R.A. Wise, Ventral tegmental site of opiate reward: antagonism by a hydrophilic opiate receptor blocker, Brain Res. 258 Ž1983. 105–108. w3x D.J. Calcagnetti, M.D. Schechter, Extinction of cocaine-induced place approach in rats: a validation of the "biased" conditioning procedure, Brain Res. Bull. 30 Ž1993. 695–700. w4x G.L. Collingridge, W. Singer, Excitatory amino acid receptors and synaptic plasticity, Trends Pharmacol. Sci. 11 Ž1990. 290–296. w5x H. Dai, R.J. Carey, The NMDA antagonist MK-801 can impair attention to exteroceptive stimuli, Beh. Brain Res. 62 Ž1994. 149– 156. w6x R.A. Goyer, Toxic effects of metals, in: C.D. Klaassen, A.O. Amdur, J. Doull ŽEds.. Toxicology, Macmillan, New York, 1986, pp. 582–635. w7x S.E. Hemby, J.E. Smith, S.I. Dworkin, The effects of eticlopride and naltrexone on responding maintained by food, cocaine, heroin and cocainerheroin combinations in rats, J. Pharmacol. Exp. Ther. 277 Ž1996. 1247–1258. w8x D.C. Hoffman, The use of place conditioning in studying the neuropharmacology of drug reinforcement, Brain Res. Bull. 23 Ž1989. 373–387. w9x P.W. Kalivas, J. Stewart, Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity, Brain Res.-Brain. Res. Rev. 16 Ž1991. 223–244. w10x C.D. Klaassen, Effect of metallothionein on the hepatic disposition of metals, Am. J. Physiol. 41 Ž1977. 101–112. w11x A.J. Lawrence, A. Michalkiewicz, J.S. Morley, K. MacKinnon, D. Billington, Differential inhibition of hepatic morphine UDPglucuronosyltransferases by metal ions, Biochem. Pharmacol. 43 Ž1992. 2335–2340. w12x P. Legendre, G.L. Westbrook, The inhibition of single N-methyl-Daspartate-activated channels by zinc ions on cultured rat neurones, J. Physiol. 429 Ž1990. 429–449. w13x G. Maranelli, P. Apostoli, P. Ferrari, Influence of smoking, alcohol, and dietary habits on blood Pb and Cd levels, Bull. Environ. Contamin. Toxicol. 45 Ž1990. 804–810. w14x R.F. Mucha, A. Herz, Motivational properties of kappa and mu opioid receptor agonists studied with place and taste preference conditioning, Psychopharmacology 86 Ž1985. 274–280. w15x J.R. Nation, D.M. Baker, M.A. Fantasia, A.E. Ruscher, D.E. Clark, Ethanol consumption and free operant avoidance performance following exposure to dietary lead, Neurotoxicol. 8 Ž1987. 561–568. w16x J.R. Nation, C.L. Livermore, G.R. Bratton, S. Schenk, Chronic cadmium exposure alters cocaine self-administration in adult male rats, Exp. Clin. Psychopharmacol. 4 Ž1996. 264–270. w17x J.R. Nation, D.K. Miller, C.L. Livermore, Chronic exposure to
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Research report
Chronic cadmium exposure attenuates the conditioned reinforcing properties of morphine and fentanyl Dennis K. Miller, Jack R. Nation
)
Department of Psychology, Texas A & M UniÕersity, College Station, TX 77643, USA Accepted 12 August 1997
Abstract Adult male rats were exposed ad libitum for 40 days to 100 ppm cadmium chloride through their diet, or an identical diet with no added cadmium. Conditioned place preference ŽCPP. was conducted in a 2-chamber apparatus in which all drugs were paired with the least-preferred side as determined on a pre-test. In Experiment 1, control and cadmium-exposed rats received 0, 0.6, 1.25, 2.5, or 5 mgrkg morphine sulfate Ži.p.. for 4 days, and vehicle only for 4 days. Control animals showed a preference for the drug-paired side at 1.25, 2.5, and 5 mgrkg while the cadmium-exposed rats showed a preference at 5 mgrkg only. In Experiment 2, rats were implanted with cannulae into the lateral ventricles and 0, 2, or 5 mg morphine sulfate was administered intracerebroventricularly Ži.c.v... An attenuation by cadmium again was observed, as control animals showed a place preference at 2 and 5 mg and cadmium-exposed animals showed preference at 5 mg only. In Experiment 3, increasing doses of the m-opioid receptor agonist fentanyl Ž0, 0.0004, 0.004, and 0.04 mgrkg. were systemically administered Žs.c.. and rats tested for CPP. While cadmium animals showed place preference only at 0.04 mgrkg, control animals showed preference at 0.0004, 0.004, and 0.04 mgrkg. These findings are discussed within the framework of metal-induced disturbance of neurochemical function andror associative processing, and the implications that such disturbances may have for drug seeking and taking. q 1997 Elsevier Science B.V. Keywords: Behavior; Cadmium; Fentanyl; Morphine; Opiate; Place preference
1. Introduction Cadmium is a widely distributed metal that selectively accrues in tobacco leaves w30x. Not surprisingly, cigarette smoking has been shown to elevate the body burden of cadmium to a level twice that of non-smokers w22,29,30x. The effects of long-term exposure to cadmium are significant as the metal is efficiently retained in the body and accumulates with age w10x. Due to the higher body burdens of cadmium in smokers, and a correlation between tobacco use and other drug taking w24x, it is important to examine the potential interactions of this heavy metal with drugs of abuse. Using an animal model, chronic cadmium exposure, at blood tissue levels that approximate smokers, has been shown to increase the intake of ethanol w18x and cocaine Abbreviations: CPP, conditioned place preference; DA, dopamine; NA, nucleus accumbens; VTA, ventral tegmental area; NMDA, Nmethyl-D-aspartate ) Corresponding author. Fax: q1 Ž409. 845-4727; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 1 3 - 5
w16x. Additionally, it has been reported recently that animals exposed to chronic cadmium show an attenuation to the locomotor decreasing effects of acute systemic morphine administration, as well as the locomotor increasing Žbehavioral sensitization. effects of morphine associated with repeated drug exposures w17x. The behavioral sensitization effects produced by repeated opioid administration appear to be mediated by an increase in activation of dopamine ŽDA. projections in the nucleus accumbens ŽNA.. This increase may derive from N-methyl-D-aspartate ŽNMDA. receptor modulation of DA pathways, andror activation of ventral tegmental area ŽVTA. m-opioid receptors that compromise the inhibitory influence of GABA interneurons, ultimately causing an increase in DA activity w9x. The established attenuation of morphine locomotor sensitization by cadmium could be due to an antagonism at any part of this proposed pathway. Cadmium blocks voltage-dependent NMDA channels w12x, decreases the stimulated release of DA w20x, and has been demonstrated to act as a competitive m-opioid antagonist w27x. It has been difficult to separate the effects of the metal on these systems using behavioral techniques. Few
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behavioral studies of cadmium toxicity have involved local applications of the metal, and given that toxicant clearance cannot be accomplished effectively in the intact animal w10x, it is unlikely that such studies would yield useful information. Regardless of the neural loci associated with cadmiumropiate interactions, the pattern of results produced by cadmium exposure has profound implications with respect to defining the abuse potential of morphine and other addictive drugs that operate via activation of opioid receptor subtypes. Along these lines, considerable research exists to demonstrate the importance of drug-related stimuli in controlling the behavior and subjective perceptions of drugs in humans, and an appropriate paradigm to study such motivational factors underlying drug use is conditioned place preference ŽCPP.. In this model, a drug is administered to the animal immediately before placement in an environment with unique contextual stimuli Žolfactory, visual, tactile.. Following several pairings of the drug and the unique context, and separate pairings of a distinctively different context and no-drug Žvehicle., the animal is tested for preference by being allowed free-choice access to the drug-paired and the vehicle-paired context. CPP is then defined by some measure of preference for one environment over another. In this regard, morphine and other opioid or non-opioid compounds have been shown to produce a robust CPP in animals that have not been exposed to exogenous chemical contamination w8x. The m-opioid receptor is believed to be influential in the reinforcing properties of opiates, and as indicated, numerous reports are available which show systemic administration of morphine w1x or the selective m-opioid agonist fentanyl w14x produce place preference. Inasmuch as cadmium acts as a m-receptor antagonist, it follows that cadmium exposure should diminish the reinforcing properties of m-opioid agonists such as morphine and fentanyl, and therein alter performance on a CPP task. In addition to the essential role of the m-receptor subtype in the development of CPP, it is established that disturbances in DA function following NA lesions by 6-OHDA w25x have parallel effects in terms of attenuating the rewarding effects of opiates. The NMDA antagonist MK-801 has also been shown to attenuate CPP, although its impact on morphine reinforcement versus associative learning has yet to be determined w28x. Because of the aforementioned disrupting effects of cadmium on NMDA and DA systems w12,20x, there is increased justification for pursuing issues relating to cadmium-related changes in the reinforcement efficacy of opioid agonists. Accordingly, employing a CPP preparation, the present report presents findings from a series of investigations on the effects of chronic dietary cadmium exposure on the conditioned reinforcing effects of the m-opioid receptor agonists morphine and fentanyl. Experiments 1 and 2 assessed the effects of chronic cadmium exposure on
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systemic Ži.p.. and intracerebroventricular Ži.c.v.. administered morphine on CPP, and Experiment 3 examined the effects on systemic Žs.c.. fentanyl on CPP.
2. Materials and methods 2.1. Apparatus Place conditioning and testing was conducted in seven 20 = 60 = 20 cm wooden shuttle boxes with wooden tilt floors. At each end of a box was a microswitch interfaced with an IBM compatible computer. A BASIC computer program was written to continuously record the number of times and duration the switch was activated through a tilt of the floor by a rat. Half the box had white walls with a smooth white floor and the other had black walls with a black sandpaper floor. For conditioning sessions, the boxes were divided into two equal-sized compartments by removable partitions. On test sessions the partitions were removed and a 20 = 10 = 5 cm wooden platform was installed 2 cm above the floor to divide the two compartments but allow free access by rats. Subjects showed a strong preference for the black side in experiments conducted before those presented here. To attenuate this preference, a 40 W light was positioned 50 cm above the black side of each apparatus. These seven lamps provided the only illumination in the room. Following each conditioning and test session the apparatus was cleaned with a mild soap solution. The apparatus was located in a sound-resistant room with a 40 dB white noise generator operating continuously. 2.2. Drugs Morphine sulfate was provided gratis by the Research Technology Branch of NIDA. For i.p. administration, the drug was dissolved in a filtered water vehicle, and the dosage is expressed as the salt. The morphine sulfate for i.c.v. administration was dissolved in a Ringer’s solution made of 120 mM NaCl, 2 mM CaCl 2 , and 2 mM NaHCO 3 in sterile water. Fentanyl ŽSigma Co.; St. Louis, MO, USA. was dissolved in a 0.9% saline vehicle and the dosage is expressed as the base from fentanyl citrate. The drug was administered s.c. 2.3. Experiment 1: systemic morphine administration 2.3.1. Subjects The subjects used in this study were 93 male SpragueDawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival at the laboratory. Body weights ranged from 175 to 199 g. Forty-six animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 47 animals received ad libitum access to an identical diet, except that it con-
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tained 100 ppm cadmium chloride. Both were specially prepared by Dyets, Inc. ŽBethlehem, PA, USA.. Continuous access to tap water was available in the home cages. Throughout the experiment, animals were double-housed in hanging polycarbonate cages. Each cage contained two members of the same exposure group Žcadmium or control diet.. The cages were located in a temperature and humidity controlled animal colony with continuous light exposure. Food consumption and body weights were recorded weekly throughout the experiment. Individual animal food consumption was measured by halving the total consumed by the cage pair. Behavioral training and testing sessions were conducted at 08:00 h. The animal holding and testing facility is approved by the Association for Assessment and Accreditation of Laboratory Animals Care International ŽAAALAC International., and all animal maintenance and research was conducted in accordance with the guidelines provided by the University Laboratory Animal Care Committee ŽULACC.. The health of the animals was monitored throughout the duration of the project by the campus veterinarian.
2.3.2. Procedures for Experiment 1 Following 40 days of pre-exposure to their appropriate control or cadmium diets, the animals began CPP conditioning and testing. On Day 1 of the study, animals were transferred from the colony to the testing room for 40 min to habituate to the sound and illumination of the room. They were not placed in the CPP apparatus. Initial biases for the white or black chamber Žpre-test. were determined on Day 2 as non-injected animals were given free access to either chamber for 15 min. On Days 3–10 the animals received one of five injections of morphine Ž0, 0.6, 1.25, 2.5, or 5 mgrkg, i.p.. on four of the conditioning days, and four vehicle Žwater. injections on the remaining four conditioning days. All injections were given at a volume of 1 mlrkg. The animal was confined to the side least preferred Ždefined as the side in which the animal spent the least amount of time on the Day 2 pre-test., 5 min after receiving a morphine injection. The animal was confined to the most-preferred chamber Ždefined as the side in which the animal spent the most amount of time on the Day 2 pre-test., 5 min following a vehicle injection. Drug and vehicle were presented on alternating days and the injection received first was counterbalanced for each dose and exposure regimen Žcadmium or control.. Animals were run in squads of seven, counterbalancing by dose and group assignment. The period of confinement for each conditioning trial was 40 min. A post-test was conducted on Day 11 following the same procedure as the Day 2 pre-test. Twenty-four hours after the post-test, animals were rendered unconscious with 60 mgrkg sodium pentobarbital Ži.p.., decapitated, and approximately 3–5 ml of trunk blood was collected.
2.4. Experiment 2: intracerebroÕentricular (i.c.Õ.) morphine administration 2.4.1. Subjects The subjects used in Experiment 2 were 45 male Sprague-Dawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival at the laboratory. Body weights ranged from 175 to 199 g. Twenty-three animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 22 animals received ad libitum access to an identical diet, except that it contained 100 ppm cadmium chloride. All other aspects of animal maintenance were as described in Experiment 1. 2.4.2. Procedures for Experiment 2 Following 40 days of pre-exposure to their appropriate cadmium or control diets, each animal was implanted stereotaxically with a unilateral guide cannula at the following stereotaxic coordinates: 1.0 mm posterior and 4.2 mm ventral from Bregma, and y1.8 mm from the midline w21x. The guide cannula was 13 mm of 22-gauge stainless steel tubing protected by a 30-gauge stainless steel obturator. Anesthesia was produced by 50 mgrkg Ži.p.. sodium pentobarbital. Following surgery, animals were housed singly through the completion of the experiment. Conditioning and testing began 1 week after surgery. Procedures for Days 1 and 2 were identical to the previous experiment. On Days 3–6 each obturator was removed and a 30-gauge injector Ž18 mm. was inserted through the outer guide cannula. After 1 min each animal was infused with 0 ŽRinger’s solution., 2, or 5 mg morphine in a volume of 1 ml. All solutions were administered by hand over a 15 s period using a microliter syringe. Immediately after receiving a drug injection the animal was confined to the side least preferred as determined on Day 2. Immediately after receiving a vehicle injection the animal was confined to the most-preferred chamber. The period of confinement was 50 min. A post-test was conducted on Day 7 following the same procedure as the Day 2 pre-test. Twenty-four hours following testing, each animal was sacrificed with pentobarbital. Approximately 3–5 ml blood was collected via a cardiac puncture and then the animal was immediately perfused with 10% formalin. Before removing the brain, 2 ml of cresyl violet ŽSigma Co.; St. Louis, MO, USA. in distilled water solution was injected through the cannula. After 10 min, the brain was removed and a transverse slice was made 1 mm posterior of the cannula site. The third ventricle was visually examined for the presence of the colored solution as an initial verification of appropriate cannulae placement. Brains were prepared in a formalin solution and 1 week later, cannula tracts were confirmed by histological preparation of 50 mm transverse slices. The behavioral data collected for each animal were used in the analysis only if the cresyl
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violet was present and the histology indicated the cannula was in the lateral ventricle. 2.5. Experiment 3: systemic fentanyl administration 2.5.1. Subjects The subjects used in Experiment 3 were 52 male Sprague-Dawley rats ŽHoltzman Co., Madison, WI, USA. that were approximately 50 days old at the time of their arrival to the laboratory. Body weights ranged from 175 to 199 g. Twenty-six animals received ad libitum access to a purified rodent diet ŽAIN-93G.. The remaining 26 animals received ad libitum access to an identical diet, except that it contained 100 ppm cadmium chloride. All other aspects of animal maintenance were as described in Experiment 1. 2.5.2. Procedures for Experiment 3 The conditioning and testing procedures for Experiment 3 were precisely as described for Experiment 1, with the exception that animals received one of four fentanyl doses Ž0, 0.0004, 0.004, and 0.04 mgrkg, s.c.. on the drug-side of the chamber during the training period ŽDays 3–10.. The fentanyl injections were given 10 min prior to placement in the chamber and the duration of confinement was 20 min. All other aspects of the study, including post-test procedures on Day 11, were as described for Experiment 1.
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sis of variance ŽANOVA. test was employed, and simple effects analyses and Newman Keuls post hoc tests were used when appropriate to determine significant differences Ž P - 0.05..
3. Results 3.1. Body weights and food consumption For each of the three experiments, there were no significant differences in body weights or food consumption according to exposure group Žcadmium or control. or dose of drug administered, as determined by ANOVAs of Group = Week and Dose = Group Ž P ) 0.05.. Body weights did increase systematically by week for all animals, and at the start of behavioral testing, body weights ranged from 440 to 530 g. Average cadmium exposure was approximately 10.0 mgrkgrday for animals receiving the adulterated diet. 3.2. Experiment 1: systemic morphine administration The animals in both exposure groups spent more time in the black chamber on the Day 2 pre-test. There were no significant differences Ž t 87 s 0.72, P ) 0.05. between cadmium-exposed Žmean s 231.1 s, S.E.M.s "22.3 s. and control Žmean s 254.4 s, S.E.M.s "23.2 s. animals with
2.6. Cadmium residues in blood Following collection of blood samples, cadmium residues in blood were measured via atomic absorption spectrophotometry w15x. 2.7. Data analysis Conditioning scores were defined by the change in time Žmeasured in s. in the drug-paired chamber from the pre-test to post-test Žtime on post-test minus time on pre-test.. A conditioned place preference occurred when the mean group conditioning score was significantly greater than the vehicle-only Ž0 mgrkg or 0 mg. group. Comparisons were made within exposure groups Žcontrol or cadmium.. For example, cadmium-morphine dose groups were compared to the cadmium vehicle-only group. Additional comparisons of group differences were made at each dose in an effort to more explicitly detail points of separation between control and cadmium-exposed animals. Four animals in Experiment 1, 7 animals in Experiment 2, and 2 animals in Experiment 3 showed a strong preference Ždefined as greater than 800 s in the 900 s pre-test. for one side over another on the Day 2 pre-test. These animals were not included in any data analysis. In addition, 2 animals in Experiment 2 were excluded due to incorrect cannulae placements. For each experiment, a Groups= Dose factorial analy-
Fig. 1. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for morphine Ži.p.. CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
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respect to the amount of time spent in the side least-preferred on the pre-test. A two-way ANOVA ŽExposure Group = Morphine Dose. was conducted on the conditioning scores yielded by Day 11 testing Žsee Fig. 1.. A significant main effect of Morphine Dose was found Ž F4,79 s 7.15, P - 0.001. and post hoc tests indicated the 5 mgrkg dose Žmean s 323.6 s, S.E.M.s "44.6 s. had greater conditioning scores than the vehicle-only Žmean s 114.2 s, S.E.M.s "32.4 s. dose. A significant main effect of Exposure Group was also found Ž F1,79 s 10.87, P - 0.01. as the control diet exposure group Žmean s 276.1 s, S.E.M.s "24.9 s. showed greater overall conditioning scores than the cadmium diet group Žmean s 182.6 s, S.E.M.s "21.3 s.. Based on a simple main effect analysis and the appropriate post hoc tests, the control animals showed significant place preference at 1.25, 2.5, and 5 mgrkg Ž F4,40 s 6.74, P - 0.05.. Within the cadmium exposure group, a significant place preference was found only at the 5 mgrkg dose as indicated by the simple main effect of dose Ž F4,39 s 2.61, P - 0.05. and post hoc tests Žsee Fig. 1.. Additional comparisons of group separation at each dose of morphine found that the control diet exposure group showed significantly greater conditioning scores than the cadmium exposure group at the 1.25 Ž t 16 s 2.82, P - 0.05. and 2.5 mgrkg Ž t 20 s 3.83, P - 0.01. doses. A marginally significant difference was seen at the 0.6 mgrkg Ž t 12 s 2.12, P s 0.055. dose. These differences in dose required to produce place preference between groups indicate a shift to the right in the dose effect curve for morphine CPP in cadmium-exposed animals. 3.3. Cadmium residues in blood For Experiment 1, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 9.16 mgrdl, S.E.M.s "3.68 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 87 s 20.01, P - 0.001.. 3.4. Experiment 2: intracerebroÕentricular (i.c.Õ.) morphine administration As in Experiment 1, both groups Žcontrol mean s 273.3 s, S.E.M.s "31.0 s; cadmium mean s 273.2 s, S.E.M.s "30.9 s. had an overall preference for the black chamber on the Day 2 pre-test. There were no differences between groups on the amount of time spent in the side least-preferred on the pre-test Ž t 34 s 0.01, P ) 0.05.. A two-way ŽExposure Group = Morphine Dose. ANOVA was conducted on the conditioning scores Žsee Fig. 2., and as in the previous experiments a significant main effect of Exposure Group was found Ž F1,30 s 8.29, P - 0.01.. Control animals Žmean s 202.9 s, S.E.M.s "45.3 s. had significantly greater conditioning scores than
Fig. 2. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for morphine Ži.c.v.. CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
cadmium-exposed animals Žmean s 47.23 s, S.E.M.s "42.3 s.. A significant main effect of Morphine Dose was also found Ž F2,30 s 3.82, P - 0.05.. Greater conditioning scores were shown at the 5 mg dose Žmean s 238.2 s, S.E.M.s "45.8 s. than vehicle-only Žmean s 48.8 s, S.E.M.s "54.5 s.. Based on a simple effects analysis and the appropriate post hoc tests a significant place preference Ž F2,17 s 4.71, P - 0.05. was shown at 2 and 5 mg for control animals. Cadmium-exposed animals also showed a significant effect of Morphine Dose Ž F2,15 s 6.78, P - 0.01. but only the 5 mg dose was different from vehicle-only. Additional comparisons of group differences at each dose found that control animals showed significantly greater conditioning scores at the 2 mg dose than cadmium-exposed animals Ž t 34 s 4.76, P - 0.01.. As with Experiment 1, a shift to the right in the dose-effect curve was seen in cadmium-exposed animals. 3.5. Cadmium residues in blood For Experiment 2, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 8.86 mgrdl, S.E.M.s "4.35 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 34 s 18.40, P - 0.001..
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3.6. Experiment 3: systemic fentanyl administration As with Experiments 1 and 2, both groups had an overall preference for the black chamber on the Day 2 pre-test. Again, there were no differences between groups Žcontrol mean s 328.6 s, S.E.M.s "16.3 s; cadmium mean s 308.9 s, S.E.M.s "23.0 s. on the amount of time spent in the side least-preferred on the pre-test Ž t 48 s 0.67, P ) 0.05.. A two-way ŽExposure Group= Fentanyl Dose. ANOVA was conducted on the fentanyl conditioning scores ŽFig. 3.. A significant main effect of Exposure Group was found Ž F1,42 s 6.75, P - 0.05., as control animals Žmean s 226.2 s, S.E.M.s "31.7 s. had greater overall conditioning scores than cadmium-exposed animals Žmean s 135.8 s, S.E.M.s "27.8 s.. A significant main effect of Fentanyl Dose was also found Ž F3,42 s 8.38, P - 0.001.. Greater conditioning scores were shown at the 0.04 mgrkg concentration Žmean s 296.6 s, S.E.M.s "27.6 s. than vehicle only Žmean s 53.7 s, S.E.M.s "26.0 s.. Based on the simple effects analysis and post hoc tests control animals showed a significant place preference at the 0.0004, 0.004, and 0.04 mgrkg dose Ž F3,22 s 4.56, P - 0.05.. A significant effect of dose also was found with cadmium-exposed animals Ž F3,20 s 5.68, P - 0.01., but only the conditioning score for the 0.04 mgrkg dose of
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fentanyl was different from the vehicle-only group. Additional comparisons of groups at each dose found that control animals showed significantly greater conditioning scores at the 0.004 mgrkg dose than cadmium-exposed animals Ž t 11 s 3.25, P - 0.01.. Thus, as with the nonselective m-opioid agonist morphine, the dose-effect curve for fentanyl was shifted to the right with cadmium exposure. 3.7. Cadmium residues in blood For Experiment 3, animals receiving the diet containing 100 ppm cadmium chloride Žmean s 8.30 mgrdl, S.E.M.s "3.68 mgrdl. had significantly greater blood cadmium levels than control animals Žmean - 0.10 mgrdl, S.E.M.- "0.10 mgrdl. receiving the unadulterated diet Ž t 48 s 20.02, P - 0.001..
4. Discussion An attenuation of the conditioned reinforcing properties of systemically administered morphine and fentanyl was observed for adult male rats exposed to chronic dietary cadmium. A complete blockade of CPP responding was not evident, however, as the cadmium-exposed animals did show a place preference, but at higher doses than controls. Additional evidence revealed that this pattern of attenuation is likely due to an antagonism by cadmium in the central nervous system, rather than a peripheral disruption of absorptionrdistribution of the drug, as a similar behavioral shift to the right in the dosereffect function was seen with morphine injected into the periphery and directly into the brain. 4.1. Systemic compared to i.c.Õ. morphine administration
Fig. 3. Mean Ž"S.E.M.. conditioning scores Žmeasured in s. for cadmium-exposed and control animals for fentanyl CPP. The numbers above the bars indicate group size. The letter ‘a’ indicates the dose is significantly different from the same dietary exposure condition Žcontrol or cadmium. vehicle-only group Ž P - 0.05.. The letter ‘b’ indicates the group showed a significantly greater conditioning score at that dose Ž P - 0.05..
The parallel findings of Experiments 1 and 2 are instructive with respect to issues relating to the locus of cadmiumropiate interactions. Because kidney and liver are target organs of cadmium toxicity w6x, physiologic dysfunction stemming from enzymatic change or other disturbances in processes linked to pharmacodynamics may have reduced the availability of the morphine at neural sites, and therein altered the impact of a given amount of the drug. Insofar as peripheral changes limited CNS penetration, the attenuation effects observed here would be defined more appropriately as metal-based changes in drug presence rather than drug potency. Because it was demonstrated in Experiment 2 that place preference resulting from i.c.v. administration of morphine was attenuated by cadmium exposure, a drug availability account of cadmium-induced attenuation of the effects of morphine is rendered less likely, at least as a sole determinant of the interaction. An alternative interpretation rests with the demonstrated effects of cadmium on neural mechanisms that play a
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modulatory role in the development and expression of the behavioral effects of morphine. In this regard, the established antagonistic actions of cadmium on DA release w20x, excitatory amino acid activation of NMDA receptors w12x, and m-opioid sites w27x gain in significance with respect to interpretive issues. Disruption of any or all of these mechanisms w2,25,28x surely could produce the patterns of attenuation observed here and elsewhere w17x. In the present case, given that cadmium did attenuate CPP following centrally administered morphine, it seems reasonable to argue that toxicant-based disturbances in transmitter activity, binding affinity, etc., are integral to the pattern of behavioral antagonism of morphine CPP. This is not to say, of course, that metal-induced changes in metabolic processes or other non-CNS events are necessarily unrelated to cadmiumropiate interactions. Rather, the findings from Experiment 2 underscore the fact that the toxic effects of the contaminant on CNS operations must be considered as a prominent component of the overall influence of cadmium on morphine sensitivity. 4.2. Fentanyl administration Related to the matter of elucidating the mechanism of action of cadmium in the brain, the data from Experiment 3 may be of value. Although it is understood that morphine preferentially acts on the m-receptor, it also is recognized that other receptor types such as the d-opioid are affected by the drug w23x, and can be influential in the development of morphine-induced CPP w26x. The findings from Experiment 3 which show that CPP produced by systemic administration of the selective m-opioid agonist fentanyl is attenuated by cadmium point to this receptor site as a critical interface in the action of the metal in the CNS. Along these lines it would be of interest to know if a selective m-agonist such as DAMGO when applied directly to a region rich in m-receptors, such as the VTA, might reverse some of the behavioral effects associated with cadmium exposure. 4.3. InterpretiÕe issues It must be acknowledged that when a biased procedure is employed, as was the case here, there are potential difficulties associated with interpreting the data. That is, an increase in time spent on the drug-paired side of the apparatus may derive either from the conditioned reward effects of the drugs, or a diminished sensitivity to the aversiveness of the least-preferred place. Whatever the underlying reasons for the change in approach behavior, in the present experiments it is clear that cadmium has a profound impact on motivational stimuli associated with morphine and fentanyl. Also, it should be recognized that the effects observed in each of the three experiments reported here may derive from challenges to associative or cognitive processing rather than biologic mechanisms. CPP is a learned phe-
nomena in which the contextual cues of the environment acquire secondary reinforcing properties via classical conditioning w3x. It has been shown that blockade of glutamate neurotransmission by MK-801 impairs attention to exteroceptive stimuli w5x, and that compromises of NMDA activity by MK-801 retard behavioral plasticity and limit associative formations in a learning context w4x. Because cadmium acts as an NMDA antagonist w12x it is reasonable to assert that the effects of the metal on opiate-induced CPP were due to alterations in learning and conditioning mechanisms rather than drug action. Still, given the fact that similar effects have been observed in behavioral preparations Žlocomotor activity. where non-associative processes dictate the response profile w17x more pharmacologic accounts of our present findings likely are preferred. 4.4. Implications Finally, it is important to establish the relevancy of the present findings for the population of humans who abuse opiates. The cadmium residue levels in blood of the metal-exposed animals in this study approximate those of pack-a-day smokers w13x. Because the overall pattern of attenuation in this report on opiate CPP and a prior motor activity study w17x are indicative of opiate subsensitivity in toxicant-exposed rats, risk factors associated with drug selection and self-administration must be addressed. Numerous investigations of drug self-administration have shown that antagonism of high doses of opiates results in increased responding w7,19x, and for opiate self-administration this translates into a health threat. The elevated drug amounts produced by behavioral antagonism may reach toxic levels even though overt symptomotology is masked. The situation is likely worse when cadmium is the antagonist, inasmuch as cadmium has been shown to alter hepatic morphine-3-glucuronide ŽM3G. production w11x and thus disturb morphine metabolism. For drug abusers using tobacco products, and this is the bulk of the sub-population w24x, this could mean an increase in the pathologic consequences of drug taking. The increased cadmium burden associated with tobacco use w22,29,30x and attendant rise in opiate self-administration, coupled with an exacerbated toxicity profile, may define a status of enhanced vulnerability to the negative effects of these psychoactive drugs.
Acknowledgements This research was supported by United States Public Health Service Grant DA07932.
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