Nr ur ophar macolog~ Vol. 20. pp. 587 to 591. 1981 0028-3908/ 81/ 060587-05802.00/ 0 Printed m Great Britain. All nghts reserved Copynght 0 1981 Perpamon Press Ltd zyxwvut CHRONIC LITHIUM ADM INISTRATION ALTERS THE INTERACTION BETW EEN OPIATE ANTAGONISTS AND OPIATE RECEPTORS IN VII/O S. AMIR and R. SIMANTOV Departments of Isotope Research and Genetics, Weizmann Institute of Science, Rehovot, Israel (Accepted 5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON January 1981) Summary-Chronic administration of lithium suppressed the jumping response of saline treated mice in the hot plate test but had no effect on rearing and grooming behaviour. Naloxone (0.15640 mg/kg) or naltrexone (0.15640 mg/kg) elevated by up to 130”,/,the frequency of the jumping response of mice fed with lithium but had little effect on the control animals. The same treatment with the opiate antagonists decreased, in a dose-dependent fashion, the rearing and grooming episodes. Large doses of naltrexone suppressed the jumping response of both normal mice and mice fed with lithium. Mice fed for 5-40 days with lithium showed a complete development of the increased jumping response to naloxone within 10 days. and somewhat less pronounced response after 40 days. Lithium levels in brain also reached a maximum after 10 days of lithium feeding. The increased in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG uiuo sensitivity to opiate antagonists of lithium-treated mice was not reflected by changes in either the affinity or the total number of opiate receptors as tested by the binding of [3H]na10xone to crude membrane preparations of whole brain. The data suggest that chronic administration of lithium alters the in oioo interaction between opiate antagonists. and possibly the endogenous opioid peptides, and the opiate receptors. The growing awareness in the last several years that endogenous opioids (enkephalins, endorphins) may play a role in the pathogenesis of psychotic states (e.g. Terenius, WahlstrGm, Lindstriim and Widerlow, 1976; Watson, Akil, Berger and Barchas, 1979, Ananth and Callanan, 1979) has prompted investigators to use opiate antagonists in the treatment of psychotic symptoms (Davis, Bunney, De Fraites. Kleinman, Van Kammen, Post and Wyatt, 1977; Emrich, Cording, PirCe, Kalling, Zerssen and Herz, 1977). It is of importance, therefore. to determine the nature and extent of the interaction between these agents and commonly used antipsychotic drugs. An interaction between endogenous opioids, opiate antagonists and the antipsychotic agents, haloperidol and chlorpromazine, has been demonstrated in several recent studies. For example, small doses of naloxone and haloperidol were found to act synergistically in blocking the apomophine-induced stereotyped behavior in rats (Moon, Feigenbaum, Carson and Klawans, 1980). Similarly, small doses of naloxone were found to potentiate the rate-decreasing effect of chlorpromazine on schedule controlled behaviour in the pigeon (McMillan, 1971). The chronic administration of haloperidol and chlorpromazine has been reported to increase the met-enkephalin content of the rat striatum (Hong, Yang, Gillin, Di Giulio. Fratta and Costa, 1979; Hong, Yang, Fratta and Costa, 1978). Finally, some neuroleptics have been Key words: psychoticism, enkephalin, naloxone, opiate dependence, lithium. 587 shown to possess binding affinity for brain opiate receptors (Clay and Brougham, 1975; Creese, Feinberg and Snyder, 1976; Somoza, 1978). Lithium salts are widely accepted as drugs of first choice in the treatment of most forms of recurrent endogenous affective disorders (Shou and Thomsen, 1975). Like haloperidol and chlorpromazine, the chronic administration of lithium chloride (LiCl) has been reported to result in an increase in the met-enkephalin content of rat striatum (Gillin, Hong, Yang and Costa, 1978). Furthermore, lithium has been shown to decrease the binding affinity of opiate agonists to brain opiate receptors in vitro (Pert and Snyder, 1974), to block the morphine-induced motor activation in mice (Carroll and Sharp, 1971) and to reduce morphine self-administration in dependent rats (Tomkiewicz and Steinberg, 1974). The effects of lithium on the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON in uiuo zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR actions of opiate antagonists have not been investigated. Opiate antagonists have been reported to facilitate the jumping response of mice in the hot plate test by interacting stereospecifically with opiate receptors in the brain (Jacob and Ramabadran, 1977, 1978; Ramabadran and Jacob. 1979). Furthermore, lithium has been reported to increase the binding affinity of opiate antagonists to brain opiate receptors under specific in vitro conditions (i.e. Pert and Snyder, 1974). In the present study the effects of chronic lithium administratioh on the pharmacological actions of the opiate antagonists naloxone and naltrexone were investigated in mice using a modified hot plate test. Alteration in the sensitivity to these antagonists in the chronically treated mice prompted the study in vitro 588 S. AMIRand R. S~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ MANTOV of whether lithium affected the number or the affinity of opiate antagonists to the brain opiate receptors. IEZI LiCl Subjects were adult male C57BL/6J mice. The animals were housed in large plastic cages (IO/cage) in a temperature- and light-regulated room (23”C, light on from 8 am-8 p.m.). Experimental animals had free access to Purina lab chow containing 0.2lzOi, LiCl (Teklad test diets, Mod TD 74280. Madison, WI). Control animals were fed regular Purina lab chow. Water was available ad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA libitum. Saline or different doses of naloxone or naltrexone RearingKmoming Jumps (Endo Laboratories, Garden City, NY) were injected Fig. 1. Tfie effect of chronic lithium ~d~inistra&ion on rearto experimental and control mice intra~r~toneaily, ing and grooming and jumps in a modified hot plate test 10min before testing. The e-ffects of the opiate antag(46”Cj in mice. Lithium was administered via the diet for onists on the responsiveness to pain were assessed by 10 days. Control animals were fed normal laboratory placing the mice on a constantly heated metal plate chow. The bars and the vertical lines represent means Lt: SEM of the behavioural measures. The asterisk indicates a (46°C) within a circular glass enclosure (15 cm diam. significant difference (t-test, P -K 0.05) from the lithium fed 20 cm high) for 5 min. The numbers of rearing and group. grooming episodes and escape attempts (iumps) displayed by the animals during the 5 min testing session grooming was noted between the saline-treated lithwere used as the dependent variables. ium fed and control mice. Two types of experiments were conducted. In the Figure 2 shows the effect of different doses of naloxfirst, mice were kept on the lithium-containing diet for one on the response patterns of lithium-fed and con10 days and the effects of different doses of naloxene and naltrexone were assessed. The second experiment trol mice in the hot plate test. Because of the large studied the effects of different lithium feeding regi- dilIerence between the jumping scores of the salinemens on the behaviour of naloxone (l~mg~g~treated injected, lithium-fed and control mice, the means of mice in the hot plate test. The amount of lithium in the rearing and grooming episodes and jumping responses of the naloxone-treated mice are expressed as the brain was determined spectrophotometrically a percentage of the mean activity of their respective according to the method of Schou (1958). All testing took place in the afternoon between 2 saline-treated baseline groups. As can be seen, naloxand 6pm. In all experiments the animals were used one produced a dose~ependent decrease in the frequency of rearing and grooming episodes of the lithonce only. ium-treated mice with the largest dose (16Omgkg) Opiate receptor binding experiments were conproducing over a 50% reduction in the magnitude of ducted as described (Simantov, 1979). Control or mice this response. In normally fed control mice naloxone fed with lithium for 10 days were decapitated and the also suppressed rearing and grooming with the largest brain minus the cerebellum was rapidly removed and placed in 30 ml of ice-cofd, 50mM Tris-HCl buffer, dose of the drug producing over 75% suppression. Contrary to its effect on rearing and grooming, nalpH 7.4 at 25°C. The brains were weighed, homogenoxone markedly elevated the frequency of the jumpized with a Politron tissue homogenizer (15sec, ing response in the lithium-fed mice with the strongest setting 4) and centrifuged for 10min at 40,ooOg. The effects (> 1300/,)seen with 40 mg/kg. In the normally pellets were then suspended in the Tris-HCl buffer fed control mice, naloxone produced only a moderate (1 mg original wet weight per 1 ml buffer) and 2ml increase in jumping frequency with the strongest effect aliquotes were incubated with 0.65-14.4 nM E3Hfna(2VA) obtained with the largest dose of the drug loxone (New England Nuclear, 25.4 Ci,/mmof) with or (160 mgfig). This moderate effect of nafoxone may be without 1 PM levaltorphan. Samples were incubated attributed to the lower hot-plate temperature (46°C) for 40min at 25” and further treated as described used in this study as compared to previous studies (Simantov. 1979). (Jacob and Ramabadran, 1977). Parts of the behavioural experiments were recently Whether these effects of naloxone reflect its opiate reported (Amir and Simantov, 1980). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE antagonistic activity had to be further tested. Figure 3 shows the effects of different doses of the opiate anRESULTS tagonist naltrexone. As with naloxone, naltrexone The chronic administration of lithium for 10 days produced a dose-dependent suppression of the rearing resulted in a marked suppression of the jumping re- and grooming response in both the lithium-treated sponse of saline-treated mice in the hot plate test ! and the control mice. Similarly, naltrexone selectively (Fig. f ). No difference in the frequency of rearing and j facilitated the jumping response of the lithium-treated 589 zyxwvutsrqpo Chronic lithium and opiate receptors r 0 f 300- :: I I I I I I I t I I I - Licl ---0 Control 1 I I 1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA I 0) 300 treated Jumps .G Jumps : 150 100 1 d- ‘6---o tn.1---o--_o &=-cco I ’ (*I) I I 0.156 0.625 I 2.5 I 10.0 I 40.0 (*Ml -I 100 \ QcO 50 t 4 LiCI-treated ---0 Control \ \ \ \ \ (*) n= E/group ’ 0.156 0 Q 14 1 0625 Naloxone(mg/kg) mice with the strongest effect (z 1.500/,)obtained with 2.5 mg/kg. In both the lithium-fed and control mice the largest dose of naltrexone strongly suppressed the jumping response relative to the response frequency seen in the respective saline-treated control groups. The effect of different lithium feeding regimens on the response to naloxone (lOmg/kg) in the hot plate test is shown in Figure 4. The behavioural data are expressed as a percentage of the mean activity of the respective lithium-fed, saline-injected mice. As can be seen, naloxone suppressed the rearing and grooming response in the hot plate test with the most pronounced effect occurring with mice receiving 10 days of lithium containing diet. The jumping response of the lithium-fed, naloxone-injected mice was markedly elevated compared to the activity seen in the respective lithium-fed, saline-treated controls and the naloxone-treated group that was not fed with lithium (zero time). The most pronounced effect of naloxone on the jumping response (> 75%) was noted in mice kept on the lithium diet for 15 days (Fig. 4). However, a 5 day lithium feeding regimen was sufficient to produce a 50% increase in the jumping response of naloxonetreated mice. At longer feeding regimens the effect of naloxone was less pronounced but with a 40 day lith- ,a-,0 ;-- 1 I J 160.0 Fig. 2. The effect of different doses of naloxone on rearing and grooming and jumps of lithium-fed (10 days), and control mice in the hot plate test. Data are presented as percentages of the means of the behavioural measures of lithium-fed or control mice injected with saline prior to testing. *Significant difference from the respective salineinjected base line group, P < 0.05; **significant difference from the respective lithium-treated groups, P < 0.05. Comparisons were made on the means of the behavioural measures using Student’s t-tests. The level of significance was corrected for multiple testing. I*.**) (.) ,0/c I 2.5 I 00 I 40.0 1 16c Naltrexone(mg/kg) Fig. 3. The effect of different doses of naltrexone on rearing, grooming and jumping of lithium-fed and contol mice. Details of the experimental procedure were the same as described in the Legend of Figure 2. ium administration, jumping in naloxone-treated miced was still over 50% higher than that of the respective lithium-fed, saline-injected control mice. Notably. as in the first experiment, lithium feeding 125 100 I I 0 5 I zyxwvutsrqponmlkjihgfedcbaZYXWV I I I IO Days I I5 !’ 20 40 Fig. 4. The effect of different lithium feeding regimens on the rearing and grooming and jumping responses of naloxone (lOmg/kg)-treated mice and on brain lithium concentrations. The behavioural data is presented as percentages of the means of the respective lithium-fed. saline-injected mice. Open circles indicate whole brain lithium concentrations in lithium-fed mice. Asterisks indicate significant difference (P< 0.05) from the respective lithium-fed. salinetreated mice. S. AMIR and R. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED SIMANTOV 590 20 40 60 80 [3~]notoxone 100 12.0 (nM) Fig. 5. Binding of [3H]naloxone to brain membrane preparations of lithium-fed mice. Five mice fed for 10 days with a lithium-containing diet (circles) were compared to 10 control mice (triangles). The binding to crude membranes prepared as described in Methods was conducted with 0.65-14.4nM [3H]naloxone without or with (solid symbols) 1 PM levallorphan. Data are average of triplicate samples for each C3H]naloxone concentration. resulted in a marked decrease in the frequency of the jumping response compared to the jumping response seen in normally fed mice. After 5 days of lithium feeding there was a 31% reduction in the mean jumping response with a maximum effect (70%) occurring following 20 days of lithium administration. Interestingly, the brain lithium peaked after 10 days of lithium feeding and no significant changes in lithium concentrations were noted following longer periods of lithium administration (Fig. 4). The increased sensitivity to the opiate antagonists in lithium-treated mice may reflect changes in the density or the affinity of the opiate binding sites to the opiate antagonists. Therefore, the binding of 0.65-14.4 nMC3H]naloxone to brain membrane preparation of control, or mice fed with lithium was tested in vitro (Fig. 5). The results show no significant changes in binding of [3H]naloxone upon lithium treatment in either low (< 1.0 nM) or high (l@-14 nM) concentrations of [3H]naloxone (Fig. 5). DISCUSSION The results of the present study show that chronic lithium administration for 5-10 days, potentiated the effect of the opiate antagonists naloxone and naltrexone on the jumping response of mice in the hot plate test. Both antagonists produced a dose-dependent biphasic effect on the jumping response, with maximal activation of the jumping at 40 and 2Smg/kg, for naloxone and naltrexone, respectively. Naltrexone was also more effective as a suppressor of the jumping response if high concentrations of the antagonists (i.e. 16Omg/kg) were used. This is in line with previous studies that indicated the longer duration of the naltrexone effect as compared to naloxone. In addition, the possibility that naltrexone in large doses may possess agonistic properties should be considered. Since the suppressive effect of high concentrations of antagonists was observed both in lithium-fed and in the control mice, further experiments are necessary to determine the specificity of this effect, including the use of the inactive isomer of the antagonist. The finding that chronic administration of lithium activates the response of mice to opiate antagonists should only be considered within the context that the lithium itself strongly suppressed the jumping response of saline-injected mice. It is, therefore, possible to argue that naloxone or naltrexone reversed the suppressive effect of lithium. That lithium attenuates the normal resporse of animals to external stimuli has been recently discussed (Johnson, 1979). This effect of lithium may be relat-r.’ at least in part, to its ability to selectively increase the level of methionine enkephalin in the striatum (Gillin et al. 1978). an area which is notable for its role in the control of motor activation. Since increased levels of the brain endogenous opioids have been reported to correlate with changes in pain responsiveness (Madden, Akil, Patrick and Barchas. 1977), it may be suggested that in mice treated chronically with lithium, the opiate antagonists unmask the effect of elevated striatal levels of methionine enkephalin. It is of interest that the increased jumping response to naloxone was fully developed within 5 days of lithium application. At this time the striatal levels of methionine enkephalin reach plateau levels (Gillin et al., 1978). However, whereas the enkephalin levels decrease to normal after prolonged treatment, the potentiated jumping response to naloxone was sustained for 40 days if lithium was continuously applied, suggesting differential mechanisms. Earlier studies have demonstrated that lithium attenuates the morphine-induced behavioural activation in mice (Carrol and Sharp, 1971). Byck (1976) and Gold and Byck (1978) have speculated that the ability of lithium to decrease the high affinity binding of opiate agonists to brain opiate receptors may account for this finding. Furthermore, these investigators argued that lithium may attenuate the binding of endogenous opioids to their receptors and that this effect may account for some of the antieuphoric effects of lithium in manic patients (i.e. Byck, 1976; Gold and Byck, 1978). Since lithium, like sodium has been reported to increase the binding affinity of opiate antagonists to opiate receptors under some in vitro conditions (Pert and Snyder, 1974). a similar mechanism underlying the effects observed here may be proposed. Although chronic lithium administration is not likely to produce any significant changes in sodium concentrations, such treatment might produce some alterations in the total cationic level within specific brain sites. This effect, in turn, may increase the binding affinity of exogenous antagonists. decrease the binding of endogenous opioid agonists, and subsequently potentiate the behavioral effects of the injected antagonists. The find- Chronic lithium and opiate receptors ina reoorted here. that chronic lithium does not change the affinity of radiolabeled antagonist, [3H]naloxone. to opiate receptors in nitro, nor the total number of opiate binding sites (as tested in saturating concentrations of C3H]naloxone). does not exclude the possibility of changes in specific brain regions in uiuo. The observed interaction between opiate antagonists and LiCl may be of clinical interest. Several recent studies have reported that naloxone produced a notable improvement of symptoms in manic patients (Janowsky. Judd. Huey, Roitman, Parker and Segal, 1978; Davis and Bunney. 1980). Others have reported beneficial effects of naloxone in schizophrenics, although negative results in both manic and schizophrenic patients have also been recorded (Janowsky, Segal, Bloom, Abrams. Guillemin, 1977; Volavka, Mallaya. Baig, Perez-Cruet, 1977). Lithium is the drug of first choice in the treatment of manic patients (Schou and Thomsen, 1975) and it has also been shown to have antipsychotic effects in schizophrenic patients (Alexander, Van Kammen and Bunney. 1979). The co-administration of small doses of lithium simultaneously with naloxone may alter the antipsychotic and antimanic effects of these agents. Yet, the interaction between low, clinically relevant levels of lithium and opiate antagonists should be studied. Y Y 591 rats treated with lithium. zyxwvutsrqponmlkjihgfedcba Proc. natn. Acad. Sci.. U.S.A. 75: 2991-2993. Gold, M. S. and Byck, R. (1978). Endorphins. lithium. and naloxone: Their relationship to pathological and drug induced manic-euphoric states, National Institute on Drug Abuse Research M onograph 19: The International Challenge of Drug Abuse (Petersen. R. C., Ed.). pp. 1922209. Rockville. Hong. J. S., Yang, H.-Y. T.. Fratta, W. and Costa. E. (1978). 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