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Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence
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Neuroscience Vol. 115, No. 2, pp. 463^469, 2002 I 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NALOXONE-PRECIPITATED WITHDRAWAL JUMPING IN 11 INBRED MOUSE STRAINS: EVIDENCE FOR COMMON GENETIC MECHANISMS IN ACUTE AND CHRONIC MORPHINE PHYSICAL DEPENDENCE B. KEST,a;b C. A. PALMESE,b E. HOPKINS,b M. ADLER,a A. JUNIb and J. S. MOGILc a
b
Department of Psychology and Center for Developmental Neuroscience, The College of Staten Island, City University of New York, Staten Island, NY 10314, USA
Neuropsychology Doctoral Subprogram, Queens College, City University of New York, Flushing, NY 11367, USA c
Department of Psychology, McGill University, Montreal, QC, Canada H3A 1B1
Abstract:Physical dependence is a widely known consequence of morphine intake. Although commonly associated with prolonged or repeated morphine administration, withdrawal symptoms can be elicited even after a single prior morphine exposure. What remains contentious is the extent to which physical dependence following acute and chronic morphine treatment is mediated by common physiological substrates and, accordingly, represent distinct syndromes. The genetic relationship between acute and chronic morphine dependence was thus presently studied by comparing mice of 11 inbred strains (129P3, A, AKR, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and SWR) for naloxone-precipitated withdrawal jumping responses using three subcutaneous morphine administration paradigms: acute (single injection) or chronic (three daily morphine injections for 4 days) injection, or chronic infusion (7 days via implanted osmotic minipumps). Although there were di¡erences in the magnitude of withdrawal jumping between the three di¡erent morphine administration paradigms, large and signi¢cant strain di¡erences were observed for each. In addition, the same strains were unusually sensitive or, conversely, altogether refractory to withdrawal jumping across all morphine treatment conditions. Overall, strain jumping means between acute and chronic dependence paradigms displayed a high degree of genetic correlation (r = 0.87^0.95). The signi¢cant correlation between chronic morphine injection and continuous morphine infusion discounts the possible confounding e¡ect of contextual learning and spontaneous withdrawal between chronic injections on the assessment of naloxone-precipitated withdrawal. Substantial heritability was also observed for acute and both paradigms of chronic dependence, with estimates ranging from h2 = 0.53 to 0.70. The present demonstration of a strong genetic correlation between physical dependence to morphine following acute and chronic treatment implies that genes associated with variable sensitivity in the two traits are the same, and is suggestive of shared physiological substrates. The data also demonstrate that the di¡erential genetic liability to morphine physical dependence begins with, and is predicted by, the ¢rst morphine exposure. I 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: opioid, tolerance, strain di¡erences.
2001) and duration of morphine exposure (Kest et al., 2001; El-Kadi and Sharif, 1994). A comparison of morphine time^e¡ect curves for jumping frequency and locomotor activity indicate that withdrawal-jumping responses are not an artifact of the stimulatory e¡ects of morphine (Smits, 1975). The di¡erences in jumping frequency accompanying precipitated withdrawal from a large variety of opiates in mice correlates well with the known physical dependence capacity of these drugs in man (Saelens et al., 1971), lending additional validity to this measure. Although physical dependence is most commonly associated with chronic morphine intake, it has also been well demonstrated that symptoms of withdrawal can be observed following even a single prior morphine exposure in a wide variety of species (Schulteis et al., 1997; Wiley and Downs, 1979; McLemore et al., 1997; Smits, 1975; Ritzmann, 1981; Kest et al., 2001), including humans (Heishman et al., 1989; Bickel et al., 1988; Heishman et al., 1990; Jones, 1980). As with chronic morphine exposure, there is a positive dose^response
Physical dependence on opioid drugs such as morphine can be inferred from multiple aversive behavioral and physiological signs following precipitation of withdrawal with narcotic antagonists such as naloxone. Among such signs, jumping is widely considered the most sensitive and reliable index of withdrawal intensity in rodents (especially mice), and is the most commonly used (El-Kadi and Sharif, 1994; Ritzmann, 1981; Smits, 1975; Saelens et al., 1971; Way et al., 1969; Miyamoto and Takemori, 1993; Kest et al., 2001). The frequency of naloxone-precipitated withdrawal jumping following chronic morphine injection shows a positive relationship with increases in morphine or naloxone dose (Kest et al.,
*Correspondence to: B. Kest, Department of Psychology (4S-223), The College of Staten Island/CUNY, 2800 Victory Boulevard, Staten Island, NY 10314, USA. Tel.: +1-718-982-4070; fax: +1718-982-3794. E-mail address: [email protected] (B. Kest). Abbreviations : NMDA, N-methyl-D-aspartate. 463
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relationship between naloxone-precipitated jumping frequency and morphine and naloxone dose after a single acute morphine injection (Kest et al., 2001; Smits, 1975). Although the withdrawal syndrome resulting from chronic morphine treatment tends to be more intense and/or of longer duration than that observed following its acute injection, the symptoms themselves are qualitatively similar (Eisenberg, 1982; Martin and Eades, 1964; Jasinski, 1977; Bickel et al., 1988; Heishman et al., 1989; Kest et al., 2001). The consistency of these ¢ndings have led many investigators to propose that acute and chronic dependence are unitary phenomena, and that common physiological mechanisms underlie both processes. An important consequence of this position is that the adaptive mechanisms underlying physical dependence after chronic morphine treatment begin with the ¢rst exposure to the drug. Others, however, doubt that dependence produced rapidly by acute morphine exposure is the same as that produced by chronic morphine intake. Converging behavioral and biochemical lines of evidence support their contention that acute and chronic dependence are distinct. They note, for example, di¡erences in the presence or temporal order of withdrawal signs after acute and chronic morphine injection in mice (Ritzmann, 1981). Furthermore, although naloxone reliably elicits jumping responses of equal magnitude in male and female mice after a variety of chronic morphine treatment schedules (Kest et al., 2001; El-Kadi and Sharif, 1994), sex di¡erences in withdrawal sensitivity and magnitude are observed after acute but not chronic morphine injection (Kest et al., 2001). Studies with antagonists of the K-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate (NMDA) excitatory amino acid receptor subtypes demonstrate their selective mediation of acute and chronic morphine dependence, respectively (McLemore et al., 1997; Marquis et al., 1991). Acute and chronic morphine injection also di¡erentially a¡ects synaptosomal Ca2þ levels (Yamamoto et al., 1978), calmodulin activity (Nehmad et al., 1982), and catecholamine utilization (Kovacs et al., 1983). The demonstration of genetic correlation of two heritable traits among isogenic (inbred) strains can be used as evidence of the existence of pleiotropic genes with a common in£uence on both traits (Hegmann and Possidente, 1981). If the traits have genes in common, then one can strongly infer their common physiology. Thus, the major aim of the present study was to compare the sensitivities of 11 inbred mouse strains on both acute and chronic morphine dependence, and to evaluate the degree of the genetic codetermination of these phenomena.
EXPERIMENTAL PROCEDURES
Subjects Adult male and female mice (n = 10^20/sex/strain) of the following inbred strains were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) for study: 129P3, A, AKR/, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and
SWR (all ‘J’ substrains). These strains are the same as those used in a previous study of morphine antinociception (Kest et al., 1999), and were at that time chosen pseudorandomly, both to ensure representation of strains of broad interest (e.g., 129, C57BL/6) and to enhance the accuracy and generalizability of genetic correlations (Hegmann and Possidente, 1981). All mice were shipped at 4^6 weeks of age and were housed four to a cage with same sex/strain mates in the College of Staten Island Animal Facility. Mice were allowed free access to food (Purina chow) and water in a temperature-controlled (22‡C) environment maintained on a 12:12-h light/dark cycle (lights on at 07:00 h). All testing was performed following an acclimation period of at least 1 week after arrival and at an age of 7^9 weeks of age. As the number of animals necessitated testing over several weeks, subjects were incompletely counterbalanced with regards to sex and strain each test day, although attempts were made to counterbalance as much as possible. All e¡orts were made to minimize animal su¡ering and only the minimum number of animals necessary for reliable statistical power was used. Drugs Both morphine sulfate, generously supplied by the Research Resources program of the National Institute on Drug Abuse (Rockville, MD, USA), and naloxone hydrochloride (SigmaAldrich, St. Louis, MO, USA) were dissolved in 0.9% physiological saline. Both drugs were administered via the subcutaneous route in an injection volume of 10 ml/kg and, for chronic morphine infusion, at a rate of 1 Wl/h. Morphine treatment Acute dependence was induced by a single subcutaneous 50 mg/kg morphine injection followed by a single naloxone dose (30 mg/kg) 3 h later. The naloxone dose and morphine^naloxone interval chosen for this and subsequent chronic dependence studies have been previously shown to yield maximal jumping, and were used in order to maximize any possible strain di¡erences (El-Kadi and Sharif, 1994; Smits, 1975; Kest et al., 2001). In the chronic dependence condition, morphine was delivered via chronic repeated injection or continuous infusion. In the former condition, mice received morphine thrice daily (09:00 h, 13:00 h and 17:00 h) for four days using a dosing schedule of 10, 20, 40 and 40 mg/kg of morphine on days 1, 2, 3 and 4, respectively. On the testing day (day 5), a ¢nal 40 mg/kg morphine dose was administered, followed by a single naloxone dose (30 mg/kg) 3 h later. In separate groups of mice receiving morphine via continuous infusion, morphine (3.4 mg/kg/24 h) was delivered for 7 days via osmotic minipumps (Model 2001, Alza, Palo Alto, CA, USA) implanted subcutaneously under oxygen/ iso£urane inhalant anesthesia. On the day of testing, pumps were removed ^ no additional morphine was given ^ and a single naloxone dose (30 mg/kg) injected 3 h later. For mice in control conditions (naloxone-control group), saline was substituted for morphine. Naloxone-precipitated withdrawal Immediately after naloxone injection, subjects were placed into individual Plexiglas observation cylinders (25U11 cm), and the frequency of jumps for each subject was tallied over the next 15 min. Among withdrawal measures, only the jumping response ^ de¢ned as the simultaneous removal of all four paws from the horizontal surface ^ was reliably observed and found to be dose-dependent in previous studies of both acute and chronic morphine dependence (El-Kadi and Sharif, 1994; Smits, 1975; Kest et al., 2001). Thus, although symptoms such as diarrhea, ptosis, wet-dog shakes, lacrimation were occasionally observed in the present study, they were excluded from analysis. Mean jump frequency per 15 min was used as the measure of dependence for each strain.
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Data analysis Two-way (sexUstrain) ANOVAs were used to compare mean jumping frequencies in all three dependence paradigms, followed by one-way ANOVAs and Student Newman^Keuls tests where appropriate. Narrow-sense trait heritability was determined by comparing the between-strain variance to the total variance. Since animals are isogenic (i.e., genetically identical) within individual inbred strains, between-strain variance provides a measure of additive genetic (‘allelic’) variation (VA ), whereas within-strain variance (‘error variance’) represents environmental variability (VE ). An estimate of narrow-sense heritability (h2 ) for each trait was obtained using the formula: h2 = VA /(VA +VE ) (Falconer and Mackay, 1996). Since strains were chosen randomly, these values are likely accurate estimates of the true trait heritabilities (Hegmann and Possidente, 1981). To assess genetic codetermination between acute and chronic dependence, correlation coe⁄cients for mean jumping frequencies were calculated using Pearson’s statistic (rp ). Strains were also ranked from smallest to highest according to jumping frequencies and subject to Spearman’s rank statistic (rs ), ensuring that correlation estimates were not unduly in£uenced by extreme scoring strains. We have also previously performed an inbred mouse strain survey of morphine analgesic tolerance where we observed large inter-strain di¡erences in the magnitude of tolerance in these same strains, as determined by decreased potency estimates derived from rightward shifts in morphine dose^ response curves (Kest et al., 2002). Strains, morphine doses, injection frequency, and general handling and testing/environmental conditions were identical to the present study of dependence. Therefore, the correlation between these two chronic morphine-induced phenotypes was also assessed. All correlations were subject to Bonferroni correction for multiple comparisons. For all statistical tests, an K criterion level of 0.05 was employed.
RESULTS
Two-way ANOVAs revealed a signi¢cant main e¡ect of strain (P 6 0.001), but not of sex or strainUsex interaction, for all three dependence paradigms. Therefore, strain distributions for all three dependence paradigms collapsed across sex are displayed in Fig. 1. Mean jump frequencies for acute dependence are presented in Fig. 1A, whereas Fig. 1B, C illustrate strain means for chronic dependence induced by chronic injection or continuous infusion, respectively. There were no signi¢cant jumping response di¡erences between strains in the naloxone-control group (data not shown). Narrow-sense heritability of these traits was estimated as h2 = 0.53 (acute dependence), h2 = 0.70 (chronic injection dependence) and h2 = 0.69 (chronic infusion dependence), respectively. These estimates are likely biased upward because of the extreme outlier status of the
Fig. 1. Acute and chronic morphine dependence in inbred mouse strains. Bars represent mean jumping frequency in the 15-min period following a single naloxone (30 mg/kg) injection given 3 h after the completion of morphine treatment. Morphine was injected once (A: acute injection : 50 mg/kg), three times daily for 4 days (B: chronic injection: 10, 20, 40, and 40 mg/kg on days 1^ 4, respectively), or continuously infused (C: continuous infusion: 3.4 mg/kg/24 h) for 7 days. Some strain names are abbreviated as follows : 129, 129P3; B6, C57BL/6; Bc, BALB/c; C3H, C3H/He. Note di¡erences in y-axis scales.
SWR strain. Even if this strain is excluded, however, heritability estimates remain moderately high, at h2 = 0.34, 0.43 and 0.53, respectively. Figure 2 presents a scatterplot matrix showing the regression of strain ranks for naloxone-precipitated withdrawal magnitude following acute and chronic morphine
Table 1. Pearson’s product-moment (rp ) and Spearman’s rank (rs , italics) correlation coe⁄cients between inbred strains means on acute and chronic morphine dependence Morphine treatment
Acute injection
Chronic injection
Continuous infusion
Acute injection Chronic injection Continuous infusion
^ 0.92 0.87
0.87 ^ 0.95
0.80 0.85 ^
Morphine was injected once (acute injection: 50 mg/kg), three times daily for 4 days (chronic injection : 10, 20, 40, and 40 mg/kg on days 1^4, respectively), or continuously infused (continuous infusion : 3.4 mg/kg/24 h) for 7 days. All correlation coe⁄cients were signi¢cant (P 6 0.01) after Bonferroni correction for multiple comparisons.
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Fig. 2. Genetic correlation of acute and chronic morphine dependence. Symbols represent inbred mouse strains arranged by their rank order of naloxone-precipitated withdrawal jumping frequency after morphine administration by acute and chronic injection or continuous infusion (1, lowest frequency ; 11, highest frequency). Spearman rank correlation coe⁄cients range from 0.80 to 0.87 (see Table 1).
treatment. The visual impressions of a correlation between responses are statistically con¢rmed by correlation coe⁄cients provided in Table 1. All pairwise correlation coe⁄cients were signi¢cant after Bonferroni correction for multiple comparisons, indicating a high degree of positive genetic correlation between acute and chronic morphine dependence. The correlation between strain rank order sensitivity to dependence and analgesic tolerance following chronic morphine injection was also signi¢cant (rs = 0.72, P 6 0.05). The outlying withdrawal responses of the SWR strain rendered the rp statistic untrustworthy in this case and thus not presented. Figure 3 presents a scatterplot matrix showing the regression of strain ranks for the two traits and illustrates their strong association.
DISCUSSION
The present study is the ¢rst to assess the frequency of naloxone-precipitated jumping in inbred mice following a single morphine injection. The data demonstrate that, similar to previous ¢ndings of chronic morphine dependence (Brase et al., 1977; Navarro et al., 1991), the magnitude of acute morphine dependence is associated with genetic variability. In addition, this is the ¢rst systematic,
multiple-strain analysis of the genetic basis of chronic dependence, and thus withdrawal magnitude for most of the strains in the present study is characterized for the ¢rst time. We were thereby able to identify several strains that are highly insensitive or all together refractory to morphine dependence (129P3, LP, SJL), and, by contrast, one strain that displayed unusual relative sensitivity (SWR). Finally, we demonstrate that substantial strain di¡erences in morphine dependence can also be elicited when a relatively small amount of morphine is delivered by continuous infusion via subcutaneously implanted osmotic minipumps. All strains considered, the magnitude of dependence was greater following chronic relative to acute morphine administration, consistent with previous quantitative comparisons of acute and chronic morphine dependence in various species including humans (Eisenberg, 1982; Martin and Eades, 1964; Jasinski, 1977; Bickel et al., 1988; Heishman et al., 1989; Kest et al., 2001). Genetic correlations By using a su⁄cient number of randomly chosen strains, the present study design should provide a valid estimate of the genetic correlation of acute and chronic dependence traits (Hegmann and Possidente, 1981). Therefore, the covariation of naloxone-precipitated
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jumping after acute and chronic morphine injection among strains suggests that common genes in£uence these traits. The demonstration of genetic correlation also implies the involvement of common (although not necessarily identical) physiological substrates, suggesting that naloxone-precipitated withdrawal jumping in mice after a single morphine exposure very likely re£ects the development of morphine physical dependence, and are not artifactual motor responses (Ritzmann, 1981). The remarkably high degree of correlation between acute and chronic dependence induced by morphine injection (rp = 0.92; rs = 0.87) attests to the resiliency of their relationship despite di¡erences in treatment duration (one injection vs. three daily injections for 4 days) and cumulative drug dosing (50 mg/kg and 340 mg/kg of morphine, respectively). Furthermore, the genetic similarity between jumping responses after acute and chronic morphine administration is not unduly in£uenced by outlier strains that can arti¢cially in£ate correlation estimates. Indeed, removing data of the unusually sensitive SWR strain from analysis only slightly reduces the rs value from 0.87 to a still signi¢cant value of 0.83. In a related manner, it is possible that the genetic relatedness between some inbred strains, thus constituting a ‘genetic family’, also contributed to the high degree of correlation between the acute and chronic injection studies. For example, 129P3, A, LP, and SJL mice all display a fair degree of genetic similarity (Atchley and Fitch, 1991; Taylor, 1972), and as a group also displayed the lowest levels of responding. Conversely, SWR mice are very much an outlier strain overall in terms of its genetic relatedness to the other strains of the present study (Taylor, 1972), and therefore, perhaps not surprisingly, displayed a uniquely sensitive phenotype. However, the strain most similar genetically to SWR mice would be those of the SJL strain (Atchley and Fitch, 1991), which were among the least sensitive of strains under study. An inspection of Fig. 2 gives no evidence that the high correlations obtained are merely an artefact of bimodal sensitivities in the 11 strains examined. Nonetheless, our ¢ndings are obviously limited to the 11 strains presently surveyed, and assumptions about the genetic commonality of acute and chronic physical dependence in other species, and even in other inbred strains, would obviously be speculative. Dependence induced by chronic morphine injection was also very highly correlated (rp = 0.95; rs = 0.85) with strain variation in naloxone-precipitated jumping responses following continuous subcutaneous morphine infusion. Chronic morphine infusion provides an important measure of reliability and validity in our assessment of dependence after chronic morphine injection for the following reasons. First, withdrawal symptoms, including jumping frequency, have been shown to depend on the method of dependence induction (Way et al., 1969; Cerletti et al., 1976). The impressive correlation between chronic injection and continuous infusion therefore demonstrates that the observed strain di¡erences are robust and not conditional on methodological parameters. Second, by allowing for the chronic delivery of drug in the absence of repeated animal handling and injection, con-
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tinuous infusion substantially diminishes the possible confounding contribution of strain di¡erences in contextual learning, which has clearly been shown to a¡ect dependence liability (Azorlosa et al., 1994; De¡nerRappold et al., 1996). Finally, spontaneous withdrawal in the absence of naloxone has been shown to emerge as early as 3 h, and last for up to as much as 24 h, following termination of morphine exposure. Given that an interval of several hours elapsed between morphine doses in the chronic injection paradigm, one cannot dismiss the possible occurrence of one or more intervening episodes of spontaneous withdrawal, thereby confounding our measurement of naloxone-precipitated withdrawal magnitude at the conclusion of the chronic morphine injection protocol. Therefore, the signi¢cant correlation of strain means and strain rank between chronic injection and continuous infusion engenders con¢dence in our assessment of inter-strain variability in dependence after chronic morphine injection. Dependence-resistant and -sensitive strains The present study identi¢ed three strains ^ 129P3, LP and SJL ^ in whom naloxone-precipitated withdrawal was relatively negligible, or altogether absent, after acute and chronic morphine administration (Fig. 1). These inbred strains thus represent potentially valuable research tools in the genetic study of morphine dependence. Since to our knowledge these strains have not been previously tested for dependence, it is not possible to compare our present ¢ndings with any existing literature. In fact, very few investigations have made use of these inbred strains, rendering speculation about the possible neurobiological substrates mediating their withdrawal resistance highly tenuous. We are similarly unable to speculate about SWR mice, which were exceptional for their unusually increased withdrawal responses relative to all other strains. Indeed, SWR mice were the most sensitive strain in every measure of dependence,
Fig. 3. Genetic correlation between morphine dependence and tolerance. Symbols represent inbred mouse strains arranged by their rank order of naloxone-precipitated withdrawal jumping frequency and reduction in morphine analgesic potency (1, lowest jumping frequency and reduction in morphine analgesic potency; 11, highest jumping frequency and reduction in morphine analgesic potency). Spearman rank correlation coe⁄cient (rs ) = 0.72.
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with mean jumping frequencies consistently between approximately 2- and 2.5-fold greater than values obtained for the most sensitive of all remaining strains. Despite the paucity of literature regarding strains of the present survey, we believe particular comment is warranted for 129P3 mice. Similar to our demonstration of withdrawal-resistance in this strain, previous studies have demonstrated that 129S6 mice (previously referred to as 129/SvEv), another 129 substrain, are resistant to morphine analgesic tolerance and naloxone-precipitated withdrawal jumping (Crain and Shen, 2000; Bilsky et al., 1999; Kolesnikov et al., 1998). Thus, reference to neurobiological characterizations of 129S6 may be of heuristic value since both substrains show similar phenotypes and are highly related (although not identical; Simpson et al., 1997) genetically. In 129S6 mice, there exists evidence that tolerance/dependence-resistance following chronic morphine treatment results from de¢ciencies in GM1 ganglioside-regulated excitatory opioid receptor-mediated functions. Indeed, the absence of tolerance is rapidly reversed by injection of low doses of exogenous GM1 ganglioside (Crain and Shen, 2000). Conversely, much higher doses of GM1 ganglioside attenuates tolerance and dependence in rats, presumably via its ability to block the translocation and activation of protein kinase C from cytosol to neuronal membranes, a biochemical pathway critical in the development of both morphine tolerance and dependence (Crain and Shen, 1998; Mayer et al., 1995). In another study, 129S6 mice were shown to possess de¢ciencies in the NMDA excitatory amino acid receptor system, and/or the biochemical cascade activating nitric oxide synthase consequent to its activation (Kolesnikov et al., 1998). The importance of the NMDA/nitric oxide signaling system in morphine tolerance and dependence has been clearly demonstrated as well (Elliott et al., 1995; Herman et al., 1995). Since tolerance and dependence are both neuroadaptive responses to chronic morphine intake, it is not surprising that these studies suggest that overlapping physiological substrates underlie both traits. Further studies are needed to determine if the compromised GM1 ganglioside and NMDA/nitric oxide signaling systems that appear to contribute to tolerance-resistance in the 129S6 mouse similarly contribute to the ability of 129P3 mice to resist morphine dependence. Nonetheless, shared physiological substrates for tolerance and dependence demonstrated by these studies implies some degree of genetic relatedness as well, and the present study indeed has presented evidence suggesting as much. Strain rank order between analgesic tolerance (Kest et al., 2002) and dependence liability following chronic morphine injection were signi¢cantly correlated (rs = 0.72). Identi¢cation of genes common to inter-individual di¡erences
in tolerance and dependence liability awaits results from current linkage analysis studies being performed independently in our laboratories for both phenotypes. Conclusions and future directions The present strain survey demonstrates that common genetic substrates underlie physical dependence to morphine following either acute or chronic morphine treatment. The data thus suggest that similar (although not necessarily identical) physiological substrates contribute to dependence liability regardless of the parameters of morphine treatment. It should be noted that although naloxone-precipitated withdrawal jumping is the most reliable and commonly used measure of physical dependence in rodents (El-Kadi and Sharif, 1994; Ritzmann, 1981; Smits, 1975; Saelens et al., 1971; Way et al., 1969; Kest et al., 2001); other indices of physical dependence have been noted. Since di¡erent neural substrates may contribute to the various signs and symptoms of the withdrawal syndrome (Koob et al., 1992), the signi¢cant correlation between acute and chronic dependence applies only to those genetic substrates of dependence that contribute to the naloxone-precipitated withdrawal jumping response. The aversive withdrawal symptomology accompanying physical dependence is regarded as a causative factor in the continued use and abuse of opioids (Jasinski, 1977). Furthermore, physical dependence remains a primary concern of physicians and has been identi¢ed as a leading cause of their undermedication of pain patients, thus hindering adequate opioid-based pain interventions (Breivik, 2001). We therefore concur with previous investigators that an examination of the entire developmental range of dependence is essential if the etiology of opioid abuse, as well as e¡ective opioid-based pain management strategies, is to be clearly elucidated. Although caution is obviously warranted when generalizing from a survey of 11 inbred mouse strains to the variation in human responses, the syntenic conservation between the mouse and human genomes suggests that study of mouse genetics can be of immense heuristic value. To this end, we have begun a linkage mapping study of dependence after chronic morphine injection using 129P3 and C57BL/6 strains as progenitors. Their consistent and relatively large di¡erences in withdrawal magnitude after acute and chronic morphine treatment should facilitate the identi¢cation of trait-relevant gene(s) mediating variable dependence liability in both traits.
Acknowledgements2Supported by PSC/CUNY Grant 62313 (B.K.) and NIH grants DE12735 and DA15191 (J.S.M.).
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Koob, G.F., Maldonado, R., Stinus, L., 1992. Neural substrates of opiate withdrawal. Trends Neurosci. 15, 186^191. Kovacs, G.L., Acsai, L., Tihanyi, A., Telegdy, G., 1983. Catecholamine utilization in distinct mouse brain nuclei during acute morphine treatment, morphine tolerance and withdrawal syndrome. Eur. J. Pharmacol. 93, 149^158. Marquis, K.L., Piesla, M.J., Muth, E.A., Boast, C.A., 1991. E¡ects of acute/chronic MK-801 on naloxone-precipitated jumping in morphinedependent mice. Soc. Neurosci. Abstr. 17, 331. Martin, W.R., Eades, C.G., 1964. A comparison between acute and chronic physical dependence in the chronic spinal dog. J. Pharmacol. Exp. Ther. 197, 385^394. Mayer, D.J., Mao, J., Price, D.D., 1995. The development of morphine tolerance and dependence is associated with translocation of protein kinase C. Pain 61, 365^374. McLemore, G.L., Kest, B., Inturrisi, C.E., 1997. The e¡ects of LY293558, an AMPA receptor antagonist, on acute and chronic morphine dependence. J. Pharmacol. Exp. Ther. 778, 120^126. Miyamoto, Y., Takemori, A.E., 1993. Relative involvement of supraspinal and spinal mu opioid receptors in morphine dependence in mice. Life Sci. 52, 1039^1044. Navarro, M., Leza, J.-C., Lizasoain, I., Lorenzo, P., 1991. In£uence of psychgenetics in opiate tolerance and abstinence in mice. Gen. Pharmacol. 22, 713^716. Nehmad, R., Nadler, H., Simantov, R., 1982. E¡ects of acute and chronic morphine treatment on calmodulin activity of rat brain. Mol. Pharmacol. 22, 389^394. Ritzmann, R.F., 1981. Opiate dependence following acute injections of morphine and naloxone: the assesment of various withdrawal signs. Pharmacol. Biochem. Behav. 14, 575^577. Saelens, J.K., Granat, F.R., Sawyer, W.K., 1971. The mouse jumping test ^ a simple screening method to estimate the physical dependence capacity of analgesics. Arch. Int. Pharmacodyn. Ther. 190, 213^218. Schulteis, G., Heyser, C.H., Koob, G.F., 1997. Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: potentiation with a second morphine exposure. Psychopharmacology 129, 56^65. Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E., Sharp, J.J., 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16, 19^27. Smits, S.E., 1975. Quantitation of physical dependence in mice by naloxone-precipitated jumping after a single dose of morphine. Res. Commun. Chem. Pathol. Pharmacol. 10, 651^661. Taylor, B.A., 1972. Genetic relationships between inbred strains of mice. J. Hered. 63, 83^86. Way, E.L., Loh, H.H., Shen, F.-H., 1969. Simultaneous quantitative assesment of morphine tolerance and physical dependence. J. Pharmacol. Exp. Ther. 167, 1^8. Wiley, J.N., Downs, D.A., 1979. Naloxone-precipitated jumping in mice pretreated with acute injections of opioids. Life Sci. 25, 797^802. Yamamoto, H., Harris, R.A., Loh, H.H., Way, E.L., 1978. E¡ects of acute and chronic morphine treatments on calcium localization and binding in brain. J. Pharmacol. Exp. Ther. 205, 255^264. (Accepted 27 June 2002)
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Neuroscience Vol. 115, No. 2, pp. 463^469, 2002 I 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NALOXONE-PRECIPITATED WITHDRAWAL JUMPING IN 11 INBRED MOUSE STRAINS: EVIDENCE FOR COMMON GENETIC MECHANISMS IN ACUTE AND CHRONIC MORPHINE PHYSICAL DEPENDENCE B. KEST,a;b C. A. PALMESE,b E. HOPKINS,b M. ADLER,a A. JUNIb and J. S. MOGILc a
b
Department of Psychology and Center for Developmental Neuroscience, The College of Staten Island, City University of New York, Staten Island, NY 10314, USA
Neuropsychology Doctoral Subprogram, Queens College, City University of New York, Flushing, NY 11367, USA c
Department of Psychology, McGill University, Montreal, QC, Canada H3A 1B1
Abstract:Physical dependence is a widely known consequence of morphine intake. Although commonly associated with prolonged or repeated morphine administration, withdrawal symptoms can be elicited even after a single prior morphine exposure. What remains contentious is the extent to which physical dependence following acute and chronic morphine treatment is mediated by common physiological substrates and, accordingly, represent distinct syndromes. The genetic relationship between acute and chronic morphine dependence was thus presently studied by comparing mice of 11 inbred strains (129P3, A, AKR, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and SWR) for naloxone-precipitated withdrawal jumping responses using three subcutaneous morphine administration paradigms: acute (single injection) or chronic (three daily morphine injections for 4 days) injection, or chronic infusion (7 days via implanted osmotic minipumps). Although there were di¡erences in the magnitude of withdrawal jumping between the three di¡erent morphine administration paradigms, large and signi¢cant strain di¡erences were observed for each. In addition, the same strains were unusually sensitive or, conversely, altogether refractory to withdrawal jumping across all morphine treatment conditions. Overall, strain jumping means between acute and chronic dependence paradigms displayed a high degree of genetic correlation (r = 0.87^0.95). The signi¢cant correlation between chronic morphine injection and continuous morphine infusion discounts the possible confounding e¡ect of contextual learning and spontaneous withdrawal between chronic injections on the assessment of naloxone-precipitated withdrawal. Substantial heritability was also observed for acute and both paradigms of chronic dependence, with estimates ranging from h2 = 0.53 to 0.70. The present demonstration of a strong genetic correlation between physical dependence to morphine following acute and chronic treatment implies that genes associated with variable sensitivity in the two traits are the same, and is suggestive of shared physiological substrates. The data also demonstrate that the di¡erential genetic liability to morphine physical dependence begins with, and is predicted by, the ¢rst morphine exposure. I 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: opioid, tolerance, strain di¡erences.
2001) and duration of morphine exposure (Kest et al., 2001; El-Kadi and Sharif, 1994). A comparison of morphine time^e¡ect curves for jumping frequency and locomotor activity indicate that withdrawal-jumping responses are not an artifact of the stimulatory e¡ects of morphine (Smits, 1975). The di¡erences in jumping frequency accompanying precipitated withdrawal from a large variety of opiates in mice correlates well with the known physical dependence capacity of these drugs in man (Saelens et al., 1971), lending additional validity to this measure. Although physical dependence is most commonly associated with chronic morphine intake, it has also been well demonstrated that symptoms of withdrawal can be observed following even a single prior morphine exposure in a wide variety of species (Schulteis et al., 1997; Wiley and Downs, 1979; McLemore et al., 1997; Smits, 1975; Ritzmann, 1981; Kest et al., 2001), including humans (Heishman et al., 1989; Bickel et al., 1988; Heishman et al., 1990; Jones, 1980). As with chronic morphine exposure, there is a positive dose^response
Physical dependence on opioid drugs such as morphine can be inferred from multiple aversive behavioral and physiological signs following precipitation of withdrawal with narcotic antagonists such as naloxone. Among such signs, jumping is widely considered the most sensitive and reliable index of withdrawal intensity in rodents (especially mice), and is the most commonly used (El-Kadi and Sharif, 1994; Ritzmann, 1981; Smits, 1975; Saelens et al., 1971; Way et al., 1969; Miyamoto and Takemori, 1993; Kest et al., 2001). The frequency of naloxone-precipitated withdrawal jumping following chronic morphine injection shows a positive relationship with increases in morphine or naloxone dose (Kest et al.,
*Correspondence to: B. Kest, Department of Psychology (4S-223), The College of Staten Island/CUNY, 2800 Victory Boulevard, Staten Island, NY 10314, USA. Tel.: +1-718-982-4070; fax: +1718-982-3794. E-mail address: [email protected] (B. Kest). Abbreviations : NMDA, N-methyl-D-aspartate. 463
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relationship between naloxone-precipitated jumping frequency and morphine and naloxone dose after a single acute morphine injection (Kest et al., 2001; Smits, 1975). Although the withdrawal syndrome resulting from chronic morphine treatment tends to be more intense and/or of longer duration than that observed following its acute injection, the symptoms themselves are qualitatively similar (Eisenberg, 1982; Martin and Eades, 1964; Jasinski, 1977; Bickel et al., 1988; Heishman et al., 1989; Kest et al., 2001). The consistency of these ¢ndings have led many investigators to propose that acute and chronic dependence are unitary phenomena, and that common physiological mechanisms underlie both processes. An important consequence of this position is that the adaptive mechanisms underlying physical dependence after chronic morphine treatment begin with the ¢rst exposure to the drug. Others, however, doubt that dependence produced rapidly by acute morphine exposure is the same as that produced by chronic morphine intake. Converging behavioral and biochemical lines of evidence support their contention that acute and chronic dependence are distinct. They note, for example, di¡erences in the presence or temporal order of withdrawal signs after acute and chronic morphine injection in mice (Ritzmann, 1981). Furthermore, although naloxone reliably elicits jumping responses of equal magnitude in male and female mice after a variety of chronic morphine treatment schedules (Kest et al., 2001; El-Kadi and Sharif, 1994), sex di¡erences in withdrawal sensitivity and magnitude are observed after acute but not chronic morphine injection (Kest et al., 2001). Studies with antagonists of the K-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate (NMDA) excitatory amino acid receptor subtypes demonstrate their selective mediation of acute and chronic morphine dependence, respectively (McLemore et al., 1997; Marquis et al., 1991). Acute and chronic morphine injection also di¡erentially a¡ects synaptosomal Ca2þ levels (Yamamoto et al., 1978), calmodulin activity (Nehmad et al., 1982), and catecholamine utilization (Kovacs et al., 1983). The demonstration of genetic correlation of two heritable traits among isogenic (inbred) strains can be used as evidence of the existence of pleiotropic genes with a common in£uence on both traits (Hegmann and Possidente, 1981). If the traits have genes in common, then one can strongly infer their common physiology. Thus, the major aim of the present study was to compare the sensitivities of 11 inbred mouse strains on both acute and chronic morphine dependence, and to evaluate the degree of the genetic codetermination of these phenomena.
EXPERIMENTAL PROCEDURES
Subjects Adult male and female mice (n = 10^20/sex/strain) of the following inbred strains were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) for study: 129P3, A, AKR/, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and
SWR (all ‘J’ substrains). These strains are the same as those used in a previous study of morphine antinociception (Kest et al., 1999), and were at that time chosen pseudorandomly, both to ensure representation of strains of broad interest (e.g., 129, C57BL/6) and to enhance the accuracy and generalizability of genetic correlations (Hegmann and Possidente, 1981). All mice were shipped at 4^6 weeks of age and were housed four to a cage with same sex/strain mates in the College of Staten Island Animal Facility. Mice were allowed free access to food (Purina chow) and water in a temperature-controlled (22‡C) environment maintained on a 12:12-h light/dark cycle (lights on at 07:00 h). All testing was performed following an acclimation period of at least 1 week after arrival and at an age of 7^9 weeks of age. As the number of animals necessitated testing over several weeks, subjects were incompletely counterbalanced with regards to sex and strain each test day, although attempts were made to counterbalance as much as possible. All e¡orts were made to minimize animal su¡ering and only the minimum number of animals necessary for reliable statistical power was used. Drugs Both morphine sulfate, generously supplied by the Research Resources program of the National Institute on Drug Abuse (Rockville, MD, USA), and naloxone hydrochloride (SigmaAldrich, St. Louis, MO, USA) were dissolved in 0.9% physiological saline. Both drugs were administered via the subcutaneous route in an injection volume of 10 ml/kg and, for chronic morphine infusion, at a rate of 1 Wl/h. Morphine treatment Acute dependence was induced by a single subcutaneous 50 mg/kg morphine injection followed by a single naloxone dose (30 mg/kg) 3 h later. The naloxone dose and morphine^naloxone interval chosen for this and subsequent chronic dependence studies have been previously shown to yield maximal jumping, and were used in order to maximize any possible strain di¡erences (El-Kadi and Sharif, 1994; Smits, 1975; Kest et al., 2001). In the chronic dependence condition, morphine was delivered via chronic repeated injection or continuous infusion. In the former condition, mice received morphine thrice daily (09:00 h, 13:00 h and 17:00 h) for four days using a dosing schedule of 10, 20, 40 and 40 mg/kg of morphine on days 1, 2, 3 and 4, respectively. On the testing day (day 5), a ¢nal 40 mg/kg morphine dose was administered, followed by a single naloxone dose (30 mg/kg) 3 h later. In separate groups of mice receiving morphine via continuous infusion, morphine (3.4 mg/kg/24 h) was delivered for 7 days via osmotic minipumps (Model 2001, Alza, Palo Alto, CA, USA) implanted subcutaneously under oxygen/ iso£urane inhalant anesthesia. On the day of testing, pumps were removed ^ no additional morphine was given ^ and a single naloxone dose (30 mg/kg) injected 3 h later. For mice in control conditions (naloxone-control group), saline was substituted for morphine. Naloxone-precipitated withdrawal Immediately after naloxone injection, subjects were placed into individual Plexiglas observation cylinders (25U11 cm), and the frequency of jumps for each subject was tallied over the next 15 min. Among withdrawal measures, only the jumping response ^ de¢ned as the simultaneous removal of all four paws from the horizontal surface ^ was reliably observed and found to be dose-dependent in previous studies of both acute and chronic morphine dependence (El-Kadi and Sharif, 1994; Smits, 1975; Kest et al., 2001). Thus, although symptoms such as diarrhea, ptosis, wet-dog shakes, lacrimation were occasionally observed in the present study, they were excluded from analysis. Mean jump frequency per 15 min was used as the measure of dependence for each strain.
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Data analysis Two-way (sexUstrain) ANOVAs were used to compare mean jumping frequencies in all three dependence paradigms, followed by one-way ANOVAs and Student Newman^Keuls tests where appropriate. Narrow-sense trait heritability was determined by comparing the between-strain variance to the total variance. Since animals are isogenic (i.e., genetically identical) within individual inbred strains, between-strain variance provides a measure of additive genetic (‘allelic’) variation (VA ), whereas within-strain variance (‘error variance’) represents environmental variability (VE ). An estimate of narrow-sense heritability (h2 ) for each trait was obtained using the formula: h2 = VA /(VA +VE ) (Falconer and Mackay, 1996). Since strains were chosen randomly, these values are likely accurate estimates of the true trait heritabilities (Hegmann and Possidente, 1981). To assess genetic codetermination between acute and chronic dependence, correlation coe⁄cients for mean jumping frequencies were calculated using Pearson’s statistic (rp ). Strains were also ranked from smallest to highest according to jumping frequencies and subject to Spearman’s rank statistic (rs ), ensuring that correlation estimates were not unduly in£uenced by extreme scoring strains. We have also previously performed an inbred mouse strain survey of morphine analgesic tolerance where we observed large inter-strain di¡erences in the magnitude of tolerance in these same strains, as determined by decreased potency estimates derived from rightward shifts in morphine dose^ response curves (Kest et al., 2002). Strains, morphine doses, injection frequency, and general handling and testing/environmental conditions were identical to the present study of dependence. Therefore, the correlation between these two chronic morphine-induced phenotypes was also assessed. All correlations were subject to Bonferroni correction for multiple comparisons. For all statistical tests, an K criterion level of 0.05 was employed.
RESULTS
Two-way ANOVAs revealed a signi¢cant main e¡ect of strain (P 6 0.001), but not of sex or strainUsex interaction, for all three dependence paradigms. Therefore, strain distributions for all three dependence paradigms collapsed across sex are displayed in Fig. 1. Mean jump frequencies for acute dependence are presented in Fig. 1A, whereas Fig. 1B, C illustrate strain means for chronic dependence induced by chronic injection or continuous infusion, respectively. There were no signi¢cant jumping response di¡erences between strains in the naloxone-control group (data not shown). Narrow-sense heritability of these traits was estimated as h2 = 0.53 (acute dependence), h2 = 0.70 (chronic injection dependence) and h2 = 0.69 (chronic infusion dependence), respectively. These estimates are likely biased upward because of the extreme outlier status of the
Fig. 1. Acute and chronic morphine dependence in inbred mouse strains. Bars represent mean jumping frequency in the 15-min period following a single naloxone (30 mg/kg) injection given 3 h after the completion of morphine treatment. Morphine was injected once (A: acute injection : 50 mg/kg), three times daily for 4 days (B: chronic injection: 10, 20, 40, and 40 mg/kg on days 1^ 4, respectively), or continuously infused (C: continuous infusion: 3.4 mg/kg/24 h) for 7 days. Some strain names are abbreviated as follows : 129, 129P3; B6, C57BL/6; Bc, BALB/c; C3H, C3H/He. Note di¡erences in y-axis scales.
SWR strain. Even if this strain is excluded, however, heritability estimates remain moderately high, at h2 = 0.34, 0.43 and 0.53, respectively. Figure 2 presents a scatterplot matrix showing the regression of strain ranks for naloxone-precipitated withdrawal magnitude following acute and chronic morphine
Table 1. Pearson’s product-moment (rp ) and Spearman’s rank (rs , italics) correlation coe⁄cients between inbred strains means on acute and chronic morphine dependence Morphine treatment
Acute injection
Chronic injection
Continuous infusion
Acute injection Chronic injection Continuous infusion
^ 0.92 0.87
0.87 ^ 0.95
0.80 0.85 ^
Morphine was injected once (acute injection: 50 mg/kg), three times daily for 4 days (chronic injection : 10, 20, 40, and 40 mg/kg on days 1^4, respectively), or continuously infused (continuous infusion : 3.4 mg/kg/24 h) for 7 days. All correlation coe⁄cients were signi¢cant (P 6 0.01) after Bonferroni correction for multiple comparisons.
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Fig. 2. Genetic correlation of acute and chronic morphine dependence. Symbols represent inbred mouse strains arranged by their rank order of naloxone-precipitated withdrawal jumping frequency after morphine administration by acute and chronic injection or continuous infusion (1, lowest frequency ; 11, highest frequency). Spearman rank correlation coe⁄cients range from 0.80 to 0.87 (see Table 1).
treatment. The visual impressions of a correlation between responses are statistically con¢rmed by correlation coe⁄cients provided in Table 1. All pairwise correlation coe⁄cients were signi¢cant after Bonferroni correction for multiple comparisons, indicating a high degree of positive genetic correlation between acute and chronic morphine dependence. The correlation between strain rank order sensitivity to dependence and analgesic tolerance following chronic morphine injection was also signi¢cant (rs = 0.72, P 6 0.05). The outlying withdrawal responses of the SWR strain rendered the rp statistic untrustworthy in this case and thus not presented. Figure 3 presents a scatterplot matrix showing the regression of strain ranks for the two traits and illustrates their strong association.
DISCUSSION
The present study is the ¢rst to assess the frequency of naloxone-precipitated jumping in inbred mice following a single morphine injection. The data demonstrate that, similar to previous ¢ndings of chronic morphine dependence (Brase et al., 1977; Navarro et al., 1991), the magnitude of acute morphine dependence is associated with genetic variability. In addition, this is the ¢rst systematic,
multiple-strain analysis of the genetic basis of chronic dependence, and thus withdrawal magnitude for most of the strains in the present study is characterized for the ¢rst time. We were thereby able to identify several strains that are highly insensitive or all together refractory to morphine dependence (129P3, LP, SJL), and, by contrast, one strain that displayed unusual relative sensitivity (SWR). Finally, we demonstrate that substantial strain di¡erences in morphine dependence can also be elicited when a relatively small amount of morphine is delivered by continuous infusion via subcutaneously implanted osmotic minipumps. All strains considered, the magnitude of dependence was greater following chronic relative to acute morphine administration, consistent with previous quantitative comparisons of acute and chronic morphine dependence in various species including humans (Eisenberg, 1982; Martin and Eades, 1964; Jasinski, 1977; Bickel et al., 1988; Heishman et al., 1989; Kest et al., 2001). Genetic correlations By using a su⁄cient number of randomly chosen strains, the present study design should provide a valid estimate of the genetic correlation of acute and chronic dependence traits (Hegmann and Possidente, 1981). Therefore, the covariation of naloxone-precipitated
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jumping after acute and chronic morphine injection among strains suggests that common genes in£uence these traits. The demonstration of genetic correlation also implies the involvement of common (although not necessarily identical) physiological substrates, suggesting that naloxone-precipitated withdrawal jumping in mice after a single morphine exposure very likely re£ects the development of morphine physical dependence, and are not artifactual motor responses (Ritzmann, 1981). The remarkably high degree of correlation between acute and chronic dependence induced by morphine injection (rp = 0.92; rs = 0.87) attests to the resiliency of their relationship despite di¡erences in treatment duration (one injection vs. three daily injections for 4 days) and cumulative drug dosing (50 mg/kg and 340 mg/kg of morphine, respectively). Furthermore, the genetic similarity between jumping responses after acute and chronic morphine administration is not unduly in£uenced by outlier strains that can arti¢cially in£ate correlation estimates. Indeed, removing data of the unusually sensitive SWR strain from analysis only slightly reduces the rs value from 0.87 to a still signi¢cant value of 0.83. In a related manner, it is possible that the genetic relatedness between some inbred strains, thus constituting a ‘genetic family’, also contributed to the high degree of correlation between the acute and chronic injection studies. For example, 129P3, A, LP, and SJL mice all display a fair degree of genetic similarity (Atchley and Fitch, 1991; Taylor, 1972), and as a group also displayed the lowest levels of responding. Conversely, SWR mice are very much an outlier strain overall in terms of its genetic relatedness to the other strains of the present study (Taylor, 1972), and therefore, perhaps not surprisingly, displayed a uniquely sensitive phenotype. However, the strain most similar genetically to SWR mice would be those of the SJL strain (Atchley and Fitch, 1991), which were among the least sensitive of strains under study. An inspection of Fig. 2 gives no evidence that the high correlations obtained are merely an artefact of bimodal sensitivities in the 11 strains examined. Nonetheless, our ¢ndings are obviously limited to the 11 strains presently surveyed, and assumptions about the genetic commonality of acute and chronic physical dependence in other species, and even in other inbred strains, would obviously be speculative. Dependence induced by chronic morphine injection was also very highly correlated (rp = 0.95; rs = 0.85) with strain variation in naloxone-precipitated jumping responses following continuous subcutaneous morphine infusion. Chronic morphine infusion provides an important measure of reliability and validity in our assessment of dependence after chronic morphine injection for the following reasons. First, withdrawal symptoms, including jumping frequency, have been shown to depend on the method of dependence induction (Way et al., 1969; Cerletti et al., 1976). The impressive correlation between chronic injection and continuous infusion therefore demonstrates that the observed strain di¡erences are robust and not conditional on methodological parameters. Second, by allowing for the chronic delivery of drug in the absence of repeated animal handling and injection, con-
467
tinuous infusion substantially diminishes the possible confounding contribution of strain di¡erences in contextual learning, which has clearly been shown to a¡ect dependence liability (Azorlosa et al., 1994; De¡nerRappold et al., 1996). Finally, spontaneous withdrawal in the absence of naloxone has been shown to emerge as early as 3 h, and last for up to as much as 24 h, following termination of morphine exposure. Given that an interval of several hours elapsed between morphine doses in the chronic injection paradigm, one cannot dismiss the possible occurrence of one or more intervening episodes of spontaneous withdrawal, thereby confounding our measurement of naloxone-precipitated withdrawal magnitude at the conclusion of the chronic morphine injection protocol. Therefore, the signi¢cant correlation of strain means and strain rank between chronic injection and continuous infusion engenders con¢dence in our assessment of inter-strain variability in dependence after chronic morphine injection. Dependence-resistant and -sensitive strains The present study identi¢ed three strains ^ 129P3, LP and SJL ^ in whom naloxone-precipitated withdrawal was relatively negligible, or altogether absent, after acute and chronic morphine administration (Fig. 1). These inbred strains thus represent potentially valuable research tools in the genetic study of morphine dependence. Since to our knowledge these strains have not been previously tested for dependence, it is not possible to compare our present ¢ndings with any existing literature. In fact, very few investigations have made use of these inbred strains, rendering speculation about the possible neurobiological substrates mediating their withdrawal resistance highly tenuous. We are similarly unable to speculate about SWR mice, which were exceptional for their unusually increased withdrawal responses relative to all other strains. Indeed, SWR mice were the most sensitive strain in every measure of dependence,
Fig. 3. Genetic correlation between morphine dependence and tolerance. Symbols represent inbred mouse strains arranged by their rank order of naloxone-precipitated withdrawal jumping frequency and reduction in morphine analgesic potency (1, lowest jumping frequency and reduction in morphine analgesic potency; 11, highest jumping frequency and reduction in morphine analgesic potency). Spearman rank correlation coe⁄cient (rs ) = 0.72.
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with mean jumping frequencies consistently between approximately 2- and 2.5-fold greater than values obtained for the most sensitive of all remaining strains. Despite the paucity of literature regarding strains of the present survey, we believe particular comment is warranted for 129P3 mice. Similar to our demonstration of withdrawal-resistance in this strain, previous studies have demonstrated that 129S6 mice (previously referred to as 129/SvEv), another 129 substrain, are resistant to morphine analgesic tolerance and naloxone-precipitated withdrawal jumping (Crain and Shen, 2000; Bilsky et al., 1999; Kolesnikov et al., 1998). Thus, reference to neurobiological characterizations of 129S6 may be of heuristic value since both substrains show similar phenotypes and are highly related (although not identical; Simpson et al., 1997) genetically. In 129S6 mice, there exists evidence that tolerance/dependence-resistance following chronic morphine treatment results from de¢ciencies in GM1 ganglioside-regulated excitatory opioid receptor-mediated functions. Indeed, the absence of tolerance is rapidly reversed by injection of low doses of exogenous GM1 ganglioside (Crain and Shen, 2000). Conversely, much higher doses of GM1 ganglioside attenuates tolerance and dependence in rats, presumably via its ability to block the translocation and activation of protein kinase C from cytosol to neuronal membranes, a biochemical pathway critical in the development of both morphine tolerance and dependence (Crain and Shen, 1998; Mayer et al., 1995). In another study, 129S6 mice were shown to possess de¢ciencies in the NMDA excitatory amino acid receptor system, and/or the biochemical cascade activating nitric oxide synthase consequent to its activation (Kolesnikov et al., 1998). The importance of the NMDA/nitric oxide signaling system in morphine tolerance and dependence has been clearly demonstrated as well (Elliott et al., 1995; Herman et al., 1995). Since tolerance and dependence are both neuroadaptive responses to chronic morphine intake, it is not surprising that these studies suggest that overlapping physiological substrates underlie both traits. Further studies are needed to determine if the compromised GM1 ganglioside and NMDA/nitric oxide signaling systems that appear to contribute to tolerance-resistance in the 129S6 mouse similarly contribute to the ability of 129P3 mice to resist morphine dependence. Nonetheless, shared physiological substrates for tolerance and dependence demonstrated by these studies implies some degree of genetic relatedness as well, and the present study indeed has presented evidence suggesting as much. Strain rank order between analgesic tolerance (Kest et al., 2002) and dependence liability following chronic morphine injection were signi¢cantly correlated (rs = 0.72). Identi¢cation of genes common to inter-individual di¡erences
in tolerance and dependence liability awaits results from current linkage analysis studies being performed independently in our laboratories for both phenotypes. Conclusions and future directions The present strain survey demonstrates that common genetic substrates underlie physical dependence to morphine following either acute or chronic morphine treatment. The data thus suggest that similar (although not necessarily identical) physiological substrates contribute to dependence liability regardless of the parameters of morphine treatment. It should be noted that although naloxone-precipitated withdrawal jumping is the most reliable and commonly used measure of physical dependence in rodents (El-Kadi and Sharif, 1994; Ritzmann, 1981; Smits, 1975; Saelens et al., 1971; Way et al., 1969; Kest et al., 2001); other indices of physical dependence have been noted. Since di¡erent neural substrates may contribute to the various signs and symptoms of the withdrawal syndrome (Koob et al., 1992), the signi¢cant correlation between acute and chronic dependence applies only to those genetic substrates of dependence that contribute to the naloxone-precipitated withdrawal jumping response. The aversive withdrawal symptomology accompanying physical dependence is regarded as a causative factor in the continued use and abuse of opioids (Jasinski, 1977). Furthermore, physical dependence remains a primary concern of physicians and has been identi¢ed as a leading cause of their undermedication of pain patients, thus hindering adequate opioid-based pain interventions (Breivik, 2001). We therefore concur with previous investigators that an examination of the entire developmental range of dependence is essential if the etiology of opioid abuse, as well as e¡ective opioid-based pain management strategies, is to be clearly elucidated. Although caution is obviously warranted when generalizing from a survey of 11 inbred mouse strains to the variation in human responses, the syntenic conservation between the mouse and human genomes suggests that study of mouse genetics can be of immense heuristic value. To this end, we have begun a linkage mapping study of dependence after chronic morphine injection using 129P3 and C57BL/6 strains as progenitors. Their consistent and relatively large di¡erences in withdrawal magnitude after acute and chronic morphine treatment should facilitate the identi¢cation of trait-relevant gene(s) mediating variable dependence liability in both traits.
Acknowledgements2Supported by PSC/CUNY Grant 62313 (B.K.) and NIH grants DE12735 and DA15191 (J.S.M.).
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