Assessment of acute and chronic morphine dependence in male and female mice

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Abstract

The present study compared male and female mice for frequency of naloxone-precipitated jumping and naloxone ED50 values, two common indices of physical dependence, following acute and chronic morphine administration. Both sexes displayed a positive dose–response relationship between acute morphine and naloxone doses and jumping frequency. There was a significant main effect of sex, with mean jumping frequencies greater in males. The naloxone ED50 estimate was also fourfold lower in males, indicating greater withdrawal sensitivity than females. Jumping frequencies were similar in male and female saline-treated control mice, discounting initial sex differences as a significant factor in the unequal magnitude and sensitivity in acute morphine dependence between sexes. In contrast, males and females displayed similar mean withdrawal jumping frequencies and naloxone ED50 values after 3 days of morphine injections. Sex difference in withdrawal jumping was also not observed when morphine treatment was increased to 7 days via daily injection or continuous subcutaneous infusion. The present study demonstrates the development of greater physical dependence in male relative to female mice following acute but not chronic morphine administration.

Introduction

Physical dependence is an unwanted side effect of morphine administration that is manifested by a characteristic withdrawal syndrome of multiple aversive behavioral and physiological signs in a wide variety of species. Withdrawal is typically observed following abrupt termination of morphine intake or precipitated by administration of a narcotic antagonist such as naloxone. Among withdrawal behaviors in rodents, jumping is widely considered the most sensitive and reliable index of withdrawal intensity and is the most commonly used El-Kadi and Sharif, 1994, Blum et al., 1976, Ritzmann, 1981, Smits, 1975, Saelens et al., 1971, Blasig et al., 1973, Way et al., 1969, Marshall and Weinstock, 1969, Fernandes et al., 1977, Miyamoto and Takemori, 1993a. A comparison of morphine time–effect curves for jumping frequency and locomotor activity in mice indicate that the withdrawal jumping response is not an artifact of the stimulatory effects of morphine (Smits, 1975). Furthermore, jumping frequencies in response to opiate withdrawal in mice correlates well with the known physical dependence capacity of these drugs in man (Saelens et al., 1971), lending validity to this measure of morphine dependence. In addition to jumping frequency, a jumping response to various doses of naloxone has also been previously used to determine a median effective dose, or naloxone ED50 estimate, for naloxone-precipitated withdrawal Way et al., 1969, Miyamoto and Takemori, 1993b, Fernandes et al., 1977, Huidoboro et al., 1963. These studies demonstrate an inverse relationship between the degree of dependence and the dose of an opioid antagonist to precipitate a critical threshold of withdrawal symptoms. Thus, whereas jumping frequency indexes the magnitude of the dependent state, the sensitivity to undergo withdrawal per se is reflected by naloxone ED50 values. Chronic morphine treatments of longer duration and/or greater cumulative dose are associated with both greater frequencies of jumping (El-Kadi and Sharif, 1994) and lower naloxone ED50 estimates Fernandes et al., 1977, Way et al., 1969.

Although physical dependence is commonly associated with chronic morphine use, symptoms of withdrawal can be precipitated by naloxone following a single exposure to morphine Schulteis et al., 1997, Wiley and Downs, 1979, McLemore et al., 1997, Smits, 1975, Ritzmann, 1981. As is observed for chronic dependence, there is a positive dose–response relationship between acute morphine or naloxone dose and magnitude of withdrawal (e.g., frequency of jumping) Smits, 1975, Fernandes et al., 1977, Ritzmann, 1981. Based on differences in the presence or temporal order of withdrawal signs, it has been suggested that acute and chronic morphine dependence are not unitary phenomena (Ritzmann, 1981). Acute and chronic morphine treatment also produces different effects on synaptosomal Ca2+ levels (Yamamoto et al., 1978), calmodulin activity (Nehmad et al., 1982), and catecholamine utilization (Kovacs et al., 1983). Furthermore, selective antagonists of the AMPA and NMDA excitatory amino acid receptor subtypes have been demonstrated to attenuate acute but not chronic morphine dependence in mice McLemore et al., 1997, Marquis et al., 1991. Collectively, these data indicate that the neural substrates contributing to dependence after acute and chronic morphine administration, as well as the adaptive physiological effects they engender, is to some extent distinct.

Morphine has been demonstrated to produce similar effects — but of different magnitude and at different doses — in males and females on a variety of acute measures, including analgesia (see review: Kest et al., 2000c), respiration (Kest et al., 1998), hypothermia Kest et al., 2000a, Quock et al., 1985, de-la-Cruz et al., 1987, activity (Li et al., 1990) and place conditioning (Cicero et al., 2000). The consequences of chronic morphine treatment may also differ between sex, as suggested by the differential development of tolerance to its analgesic Kest et al., 2000b, Craft et al., 1999, Badillo-Martinez et al., 1984 and hypothermic (Kest et al., 2000a) effects in male and female rodents. Although the mechanism underlying sex differences has been the subject of many investigations (see review: Kest et al., 2000c), a cogent explanation of these differences that incorporates the documented contribution of widely disparate variables has yet to emerge. Centrally located pharmacodynamic processes have been implicated since sex differences on behavioral measures are not simply related to sex differences blood levels of morphine and its metabolites in rats (Cicero et al., 1997) and humans (Sarton et al., 2000), but also persist following acute and chronic CNS morphine microinjection Kest et al., 1999, Boyer et al., 1998, Krzanowska and Bodnar, 1999. Despite evidence for sex differences in morphine effects on several measures, studies comparing males and females on morphine dependence are relatively scarce. In mouse studies, sex differences in dependence were not observed after 3 or 7 days of morphine treatment Blum et al., 1976, El-Kadi and Sharif, 1994. Studies in rats, by contrast, report greater dependence in females than males after 5 and 6 days of multiple daily morphine injections Craft et al., 1999, Ali et al., 1995. However, only limited conclusions regarding sex differences in morphine dependence may be drawn from these studies for the following reasons. First, and perhaps most importantly, sex comparisons in all of the above studies were made using only a single morphine and naloxone dose. The absence of dose–response data is of concern since the magnitude of withdrawal symptoms is morphine and naloxone dose-dependent Gellert and Holtzman, 1978, Way et al., 1969, Marshall and Grahame-Smith, 1971, Blasig et al., 1973, Smits, 1975, Fernandes et al., 1977, Ritzmann, 1981, Miyamoto and Takemori, 1993a. Single doses also preclude calculation of a naloxone ED50 estimate, and thus possible sex differences in morphine withdrawal sensitivity are unknown. Lastly, despite the demonstration that acute and chronic morphine treatment produces different physiological adaptations Yamamoto et al., 1978, Kovacs et al., 1983, Nehmad et al., 1982 and each elicits dependence that is mediated by distinct neurochemical substrates McLemore et al., 1997, Marquis et al., 1991, males and females have only been compared for chronic morphine dependence. Thus, the present study compared male and female mice for naloxone-precipitated jumping frequency and naloxone ED50 estimates in both acute and chronic dependence models using a range of morphine and naloxone doses.

Section snippets

Subjects and drugs

Adult male and female CD-1 mice (Charles River, Wilmington, MA) were housed four to a cage with same-sex littermates and maintained on a 12/12 h light/dark cycle in a temperature-controlled environment with unrestricted food and water. Animals were housed at least 1 week prior to testing, which was conducted near midphotophase to minimize circadian fluctuations in morphine sensitivity (Kavaliers and Hirst, 1983). Each dose of every condition was comprised of at least 10 mice/sex and each mouse

Acute dependence

As illustrated in Fig. 1, acute morphine injection precipitated jumping in a dose-dependent manner following naloxone (50 mg/kg) in both sexes (P<.001). There was also a significant (P=.01) main effect of sex, with greater mean jumping frequencies, indicative of greater dependence, in males relative to females across morphine doses (Fig. 1). There was no significant interaction between morphine dose and sex. There was no sex difference in naloxone-precipitated jumping frequencies in

Discussion

The present study demonstrates naloxone-precipitated withdrawal jumping in male and female mice after both acute and chronic morphine treatments. Jumping frequencies in the two treatment conditions displayed a positive dose–response relationship with morphine across a range of doses. Jumping was similarly dose-dependently increased with naloxone doses as high as 50 mg/kg. In CD-1 mice, naloxone doses as high as 50 mg/kg yield consistent and reliable levels of withdrawal jumping McLemore et al.,

Acknowledgements

We thank the Drug Supply System of the National Institute on Drug Abuse for supplying morphine sulfate. Supported by grants from the CSI/IBR Center for Developmental Neuroscience and PSC/CUNY.

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