Drug and Alcohol Dependence and Withdrawal Models

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1 Drug and Alcohol Dependence and Withdrawal Models John C. Crabbe, PhD Department of Behavioral Neuroscience Oregon Health & Science University, Portland, OR

3 Drug and Alcohol Dependence and Withdrawal Models 51 Introduction Dependence on alcohol and other psychoactive drugs is defined clinically by a spectrum of symptoms that include biological disturbances and, prominently, behavioral abnormalities as well. A diagnosis of substance dependence according to DSM-IV TR requires tolerance (attenuation of drug effect), withdrawal (which is thought to result from physical dependence on the drug and is defined by physical and psychological symptoms after drug-taking ceases), and compulsive drug taking. The National Institute on Alcohol Abuse and Alcoholism ( further differentiates compulsive alcohol drinking into craving versus continued drinking despite the emergence of adverse medical consequences and loss of control over drinking. For either diagnosis, drug-taking begins to intrude into work and personal relationships. These disorders are extraordinarily complex, and it is clear that there is no single behavioral or neurobiological model system that captures the entirety of drug and alcohol dependence. Neither is there any single genetic animal model of drug dependence or alcoholism none can capture the full range of disruption wreaked on humans by excessive drug taking (1). An additional complexity is the behavioral (and neurobiological) specificity of drug effects. Humans abuse a wide range of drugs from many broad pharmacological classes, including sedative-hypnotics, opiates, psychostimulants, mixed stimulant-depressant cholinergic drugs, nicotine, NMDA antagonists like phencyclidine, hallucinogens, organic solvents, steroid hormones, and, increasingly, nutriceuticals with unknown amounts of unknown substances. These compounds exert widely divergent effects in mice as well as humans, both acutely and chronically, and it is no surprise that the symptoms of withdrawal from LSD are different from those of alcohol. Furthermore, chronic administration of some drugs (e.g., depressants, opiates) leads to the development of tolerance to their sedative effects (2). On the other hand, psychostimulants generally do not reduce behaviors in mice, but stimulate them, and chronic administration leads to enhanced responding (sensitization) rather than tolerance (3). To further increase the complexity, mice respond to low doses of most depressants and opiates with psychomotor stimulation, and this response also sensitizes with chronic drug administration (4). Consequently, researchers modeling drug dependence in mice typically elect to model selected features of the disorder. Furthermore, the specific behaviors chosen for modeling are oriented toward the primary drug of interest s spectrum of effects. For example, withdrawal from alcohol and other sedative-hypnotics in humans is characterized by many symptoms, including (but not limited to): CNS hyperexcitability, even seizures in extreme cases; widespread autonomic dysregulation; anxiety and restlessness; nausea; sleeplessness; and depression. Withdrawing mice also show the CNS hyperexcitability, anxiety-like, and depression-like behaviors (5). In addition to withdrawal, specific aspects of mouse drug responses that have been behaviorally modeled include: the initial response to the drug; the neuroadaptation that occurs with chronic drug administration, whether reduced (tolerance) or increased (sensitization); withdrawal symptoms when the drug is removed; and drug reinforcement, which may be either positive or negative. Drug reinforcement is most often modeled by allowing mice to self-administer the drug (4). Although not discussed here, a variety of behavioral tendencies thought to be predictive of drug abuse risk are also studied, including impulsivity, novelty-seeking, hyperactivity, and altered reactivity of the stress axis (1). This rather long-winded introduction serves primarily to make the point that a one-hour talk cannot cover the spectrum of drug dependence phenotypes studied in mice. In this introduction, I have chosen to introduce the topic and its complexities and have selected a couple of examples to illustrate how careful phenotyping can clarify behavior-neurobiology relationships. In the talk, I will also provide examples of how uncritical phenotyping can lead one astray, and I will generally avoid topics addressed by other speakers, even though they are prominent in studies of drug dependence (e.g., anxiety-like behavior and learning and memory tasks). I will also discuss alternative versions of selected behavioral tasks and their interchangeability. Finally, because my interest is in assessments of genetic components of risk, my talk will use genetic mouse models and attempt to describe how phenotypic assessment of genetically-influenced individual differences should be approached with a sharp eye toward avoiding overinterpretation. The examples discussed in this introduction are alcohol self-administration and withdrawal severity, and their genetic relationship. Alcohol Withdrawal Nearly all studies of alcohol withdrawal in mice focus on one or two behaviors seizure (or convulsion) susceptibility (or severity), and anxiety-like behavior. Dependence on alcohol is typically induced in one of two ways. In one procedure, mice are offered a liquid diet containing alcohol as a primary source of calories, while control groups are pairfed an equivalent volume of diet with sucrose replac- NOTES

4 52 Mouse Behavioral Phenotyping NOTES ing the ethanol-derived calories. After about a week, if the diet is removed, mice will display alcohol withdrawal signs (6). The other widely employed method involves administering alcohol as a vapor. Mice are placed into chambers where they inhale the vapor, and controls are similarly treated but inhale air. While one might think that simply offering mice water with a percentage of alcohol as their only source of fluid would be the usual method, this generally leads to substantial weight loss and it is felt that this is not an ideal method for studying alcohol dependence per se. The vapor inhalation method was used in the pioneering studies that showed that susceptibility to alcohol dependence had a genetic component. Dora Goldstein exposed mice to ethanol vapor for 72 hours and scored the severity of withdrawal using handling-induced convulsion (HIC) (7). This novel behavioral index, which ranges from postural adjustments to tonicclonic convulsions, looks like the behavioral manifestation of a seizure, although it has not been electrographically characterized. In a series of elegant experiments using this index, she showed that the severity of withdrawal depended on both dose of ethanol and duration of exposure (8). By systematically mating high scorers and low scorers, a method known as selective breeding, she showed that withdrawal severity differed in their offspring, showing that withdrawal susceptibility was partially inherited (9). Since this pioneering study, many subsequent studies with inbred strains of mice have also shown a genetic influence on ethanol-induced withdrawal severity (10,11). Members of an inbred strain are genetically identical to each other, and homozygotes, at each gene, but the specific allele they all possess was determined by chance during the process of inbreeding. When strains are compared in a carefully controlled environment, any differences among strains provide evidence for genetic influence on the trait measured. Using a modification of the HIC index that allowed the detection of withdrawal severity with more sensitivity, we showed that withdrawal (increased HIC scores) could be seen after a single injection of ethanol (12). This had first been noted as a reduction in the threshold to induce seizures with the convulsant drug, pentylenetetrazol (13). Susceptibility to acute withdrawal reactions, (seen after administration of a single high dose of a drug) is also seen after barbiturates and benzodiazepines, as well as a number of other depressants, and there is a substantial genetic commonality among mouse strains in susceptibility to these effects (12,14) Interestingly, even though the same behavioral assay, the HIC, was used to index both traits, we found that susceptibility to acute pentobarbital and alcohol withdrawal were genetically very similar to each other, but that chronic alcohol withdrawal severity (after chronic inhalation) was only partially genetically overlapping in its control (15).This finding was enabled by the development of a very sensitive behavioral assay. Alcohol Reinforcement Nearly all studies of alcohol reinforcement in mice use either the willingness of mice to self-administer alcohol orally when offered versus water, or the conditioned place preference paradigm. Since the original demonstration that mice of the C57BL/6 inbred strain preferred to drink ethanol solution over water, while those of the DBA/2J strain avoided them (16). several gene mapping studies have focused on different measures of ethanol self-administration. Multiple studies have used populations derived from these two inbred mouse strains, and the locations of the genes affecting alcohol preference reported across studies are highly replicable (17). The differences among other inbred strains have been studied in many laboratories and they, too, are highly replicable. Interestingly, much as acute and chronic ethanol withdrawal severity were found to be somewhat but not completely genetically co-determined, different measures of alcohol preference seem to show somewhat different patterns of genetic control. For example, in the alcohol acceptance test, rather than offering each animal a choice between an alcohol and a water bottle, mice are first deprived of water and then offered a single bottle of dilute alcohol and their intake assessed. The genetic control (pattern of strain differences) modulating this behavior are rather different from that affecting preference (18). The Relationship between Alcohol Withdrawal Severity and Preference Individual genes almost never affect a single behavioral response; rather, their effects are seen in many behaviors, a condition termed pleiotropy. Inbred mouse strains that prefer alcohol solutions tend to be those that show mild withdrawal severity using the HIC when the drug is removed. As should be apparent from the above discussion of each of these behaviors separately, each can be measured in multiple ways. Many studies have used many different genetic models (inbred strains, recombinant inbred strains, selectively bred lines) as well as different behavioral endpoints (acute alcohol withdrawal, chronic alcohol withdrawal, alcohol preference and acceptance assessed through a variety of procedures).

5 Drug and Alcohol Dependence and Withdrawal Models 53 It is interesting that a robust pattern of negative genetic association characterizes these studies. The genetic correlation between withdrawal and preference has been estimated to be as high as r = -.65 across 15 inbred strains, but is substantial across many other conditions as well (19). It is also interesting that rat lines selectively bred to drink high versus low amounts of ethanol showed this pattern of results as well. Alcohol preferring rats showed minimal withdrawal after an acute, high-dose intragastric treatment with ethanol, while alcohol avoiding rat lines showed withdrawal signs (20). This is a case where the subtleties of the behavioral assay seem not to be so marked as to undermine ascertainment of a fundamental biological relationship. One could speculate that rodents encountering alcohol in the wild have occasionally overindulged and become intoxicated. If such intoxication led to a selective advantage, natural selection could account for the underlying relationship. However, in the laboratory studies to date that negatively link withdrawal and preference, rodents have not become intoxicated voluntarily, but have had alcohol forced on them, so this sort of evolutionary explanation is highly speculative. Summary Rodents will self-administer most drugs orally, but the earlier ascendancy of the rat in neuroscience has led to the widespread belief that only intravenous self-administration of a drug of abuse truly reflect the reinforcing effects of the drug. On the other hand, some alcohol researchers have argued strenuously that the only meaningful index of alcohol s reinforcing effects is the two-bottle preference test for raw alcohol in tap water. Raw alcohol in tap water is unappealing, to humans and to most mice, and is not how humans learn to drink, but this prejudice has hindered the development of other, alternative selfadministration methods. There is no single method with mice that will index reinforcement, or withdrawal, for all drugs of abuse. Neither is there any single behavior that can serve as a necessary and sufficient index of dependence. In the talk, we will discuss several other measures used to index various responses to drugs of abuse, but the underlying message is that multiple measures are necessary to model disorders as complex as drug dependence (21). Acknowledgments Supported by a grant from the Department of Veterans Affairs, and by A12714, AA10760, AA13519, AA13478, AA06243, DA 05828, and DA References 1. J.C. Crabbe, Annual Review of Psychology 53, (2002). 2. H. Kalant, Alcohol Clin.Exp.Res. 22, (1998). 3. G.I. Elmer, D.A. Gorelick, S.R. Goldberg, R.B. Rothman, Pharmacol Biochem.Behav. 53(3), (1996). 4. C.L. Cunningham and T.J. Phillips, in Molecular Biology of Drug Addiction, R. Maldonado, Ed. (Humana Press, Inc., Totowa, NJ, 2003). 5. H.J. Friedman, in Alcohol Tolerance and Dependence, H. Rigter and J.C. Crabbe, Eds. (Elsevier/North-Holland Biomedical Press, Amsterdam, 1980),chap L.D. Snell, G. Szabo, B. Tabakoff, P.L. Hoffman, Journal of Pharmacology and Experimental Therapeutics 279(1), (1996). 7. D.B. Goldstein and N. Pal, Science 172, (1971). 8. D.B. Goldstein, J.Pharmacol.Exp.Ther. 180, (1972). 9. D.B. Goldstein, Nature 245, (1973). 10. P.J. Griffiths and J. M. Littleton, Br. J. Exp. Pathol. 58, (1977). 11. J.C. Crabbe, J Pharmacol Exp Ther 286, (1998). 12. J.C. Crabbe, C.D. Merrill, J.K. Belknap, Journal of Pharmacology and Experimental Therapeutics 257:2, (1991). 13. D.G. McQuarrie and E. Fingl, J Pharmacol. Exp.Ther. 124, (1958). 14. P. Metten and J.C. Crabbe, Pharmacol Biochem Behav 63, (1999). 15. C. Fehr, J.K. Belknap, J.C. Crabbe, K.J. Buck, J Neurosci (2002). 16. G.E. McClearn and D.A. Rodgers, Quarterly Journal of Studies on Alcohol 20, (1959). 17. J.K. Belknap and A.L. Atkins, Mamm Genome 12, (2001). 18. L.A. Rodriguez, R. Plomin, D.A. Blizard, B.C. Jones, G.E. McClearn, Alcohol Clin.Exp.Res. 18(6), (1994). 19. P. Metten et al., Mamm Genome 9, (1998). 20. J.A. Chester, A.M. Blose, J. C. Froehlich, Alcohol Clin.Exp.Res. 27, (2003). 21. D. Wahlsten, N.R. Rustay, P. Metten, J.C. Crabbe, Trends in Neurosciences 26, (2003). NOTES

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