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The use of transgenic mice to study addictive behavior
Clinical Neuroscience Research 3 (2003) 325–331 www.elsevier.com/locate/clires
The use of transgenic mice to study addictive behavior Rainer Spanagela,*, Carles Sanchis-Seguraa,b a
Department of Psychopharmacology, Central Institute of Mental Health, J 5, D-68159 Mannheim, Germany b Departament de Psicologia Ba`sica, Clı´nica i Psicobiologia, Universitat Jaume I, Castello, Spain
Abstract Drug addiction is a behavioral disorder which arises in some individuals after repeated drug use. In these individuals, the search and consumption of the drug becomes compulsive and takes place at expense of all other vital activities, resulting in a complete loss of social compatibility (e.g. loss of partner and friends, loss of job, crime). From this definition two main consequences can be obtained. First, addiction is not a direct and inevitable consequence of drug use, a fact that suggests the existence of identifiable factors that can contribute to the vulnerability to addiction. Second, it is also apparent that addiction cannot be fully modeled in preclinical animal models. However, some specific features of addiction such as the existence of relapses even after a long period of abstinence can be successfully studied in different animal models. Moreover, different tests which do not directly reproduce any behavioral features of addictive behavior (e.g. locomotor sensitization) have been proved to have a high heuristic value in unraveling the neurobiological changes associated in the transition that leads from a casual use of a drug to the addictive state. In the present review we summarize the studies devoted to identify genetic factors that could result in a higher susceptibility to addiction by using genetically engineered mice in the preclinical model and tests available. From this overview it is concluded that, compared to the number of studies about drug self-administration, the use of transgenic mice in addictionrelated models has been rather scarce and should be further encouraged. q 2003 Elsevier B.V. All rights reserved. Keywords: Mouse; Knockout; Addiction; Sensitization; Relapse
1. Introduction Drug addiction is defined as a syndrome in which drug use (e.g. psychostimulants, opiates, alcohol) pervades all life activities of the user. The life becomes governed by the drug and the addicted patient can lose social compatibility (e.g. loss of partner and friends, loss of job, crime). Behavioral characteristics of this syndrome are compulsive drug use, craving and chronic relapses which can occur even after years of abstinence. How can addictive behavior be measured in laboratory animals? Some years ago we had an expert meeting on animal models of addiction. In the course of the discussion, it became clear that terms such as ‘compulsive drug use’ or ‘craving’ are difficult to transfer to laboratory animals and that only some aspects of addictive behavior can be studied in laboratory animals. However, one phenomenon which might give new insights into drug craving and can easily be studied in mice is drug-induced sensitization. Therefore, numerous studies using transgenic mice have been * Corresponding author. Tel.: þ49-621-170-3833; fax: þ 49-621-1703837. E-mail address: [email protected] (R. Spanagel). 1566-2772/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1566-2772(03)00094-X
published on drug-induced sensitization in the past decade [1]. Here we will present an overview of these studies. Other aspects of addictive behavior, such as relapse behavior, can be studied more directly in laboratory animals. The standard procedure used in most laboratories working on relapse behavior is the operant reinstatement model of drug-seeking behavior [2]. This model can be used to study relapse to all kind of drugs. However, other models have also been used to study relapse behavior in laboratory animals. In the second part of this review we will briefly describe these models on relapse behavior and their special application to mice. Then we will discuss studies using these procedures to examine transgenic mice for the elucidation of genes involved in relapse behavior.
2. Drug-induced sensitization in mice The term sensitization refers to an increase in a response after the repeated occurrence of the stimuli that promotes it. Sensitization in drug abuse research has been mainly studied in respect to locomotor activity. Thus the ability of addictive drugs to increase locomotion, after an acute administration,
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is progressively enhanced when drug exposure is repeated. This so-called psychomotor sensitization is a very robust phenomenon that has been observed across several species including mice. Although there are variations among sensitization protocols for mice, to achieve this effect, any sensitization protocol should use a route of administration with a fast onset of drug effect (usually intraperitoneal or intravenous injections are used) and the drug has to be given intermittently. Moreover, sensitization is stronger when the dose is higher or when escalating doses are administered [3,4]. Some drugs such as psychostimulants [5] and morphine [6] can even trigger a sensitized response after a single pre-exposure if the dose used was high enough. Finally, although sensitization is in essence a nonassociative process and it can be triggered independently of any environmental cue, the context can play a major role in the development and expression of sensitization [7,8]. Thus a higher degree of sensitization is usually observed when drug injections are administered in a context different from the home cage and subjects fail to express sensitization when they are challenged in a different context where they have never experienced the drug [9,10]. It has further been proposed that the incentive salience attributed to an initially neutral stimulus is progressively increased when this stimuli is contingent and repeatedly associated to the drug administration, therefore leading to an ‘incentive sensitization’ [8]. In this regard, the sensitization process has become one of the most commonly proposed mechanisms to explain the transition from a regular pattern of voluntary drug intake to compulsive drug-seeking and taking behavior [8,11]. However, to test the occurrence of this phenomenon a very sophisticated experimental approach is required in order to rule out other confounding processes (i.e. changes in the hedonic value of the drug). For this reason, no standard protocols to test incentive sensitization are currently available and those used till now have been only applied to rats [12,13] since the acquisition of complex contingencies related to these tests cannot be easily learned by mice. Therefore, although it is clear that the incentive sensitization may provide a more direct link to the core features of addictive behavior, psychomotor sensitization in mice has been used far more in the past.
3. The use of transgenic mice in drug-induced sensitization studies It is suggested that psychomotor and incentive sensitization are at least partially mediated by the same neural circuitry, i.e. nucleus accumbens; therefore, both phenomena are useful behavioral manifestations to assess neuroplastic changes of this neural system in relationship to drug-seeking behavior. Most of the studies about druginduced sensitization are related to repeated exposure to
psychostimulants, since these drugs act primarily on dopaminergic neurons and produce a more robust sensitization than other addictive drugs. Therefore, in this paragraph we will focus on psychostimulant-induced sensitization. It has been demonstrated that psychostimulants potentiate dopamine signaling in the mesocorticolimbic system and this effect is progressively enhanced as the administration of these drugs is repeated [14 – 16]. Thus, it has been suggested that transient increases in somatodendritic dopamine release and spontaneous firing activity of dopamine neurons in the ventral tegmental area (VTA) could be related with the induction of behavioral sensitization, whereas long-lasting alterations within dopamine neuronal terminal fields could be responsible for its expression [17]. The exact nature of these changes is not completely understood, but changes in several neural substrates involved in dopamine neurotransmission have been described, and in the last years transgenic mice have been used to confirm the involvement of these molecular components in the development/expression of psychostimulant-induced sensitization. Among these changes, the role of dopamine receptors in amphetamine or cocaine-induced sensitization has been extensively studied [18]. Thus, it has been demonstrated that D1-dopamine receptor knockout mice display a reduction in their locomotion after an acute injection of cocaine and also an attenuated sensitization response to cocaine [19]. Surprisingly, these mice show normal sensitization to amphetamine [20,21]. On the other hand, after repeated psychostimulant administration there is reduction in the sensitivity of D2-dopamine autoreceptors that could contribute to enhanced dopamine neurotransmission in the nucleus accumbens [17]. However, although this effect could play a role in the development of sensitization, the contribution of this effect to the expression of sensitization seems very unlikely because alterations of D2-dopamine autoreceptors are transient in nature compared to the persistence of the behavioral manifestation of drug-induced sensitization. In agreement with this suggestion, it has been demonstrated that the D2-dopamine receptor knockout mice display normal locomotion and sensitization after acute and chronic cocaine administration respectively [22]. Conversely, D3-dopamine receptor knockout mice display an enhanced response to an acute challenge with cocaine [23] but exhibit a normal sensitized response after repeated injections of this drug [24]. Finally, the D4-dopamine receptor knockout mice show an enhanced response to an acute challenge with cocaine or metamphetamine [25,26] but their response to repeated injections of these drugs have not yet been evaluated. It is also suggested that alterations in monoamine transporter systems – which are the primary site of action of psychostimulants – are involved in the development and expression of psychostimulant-induced sensitization [27]. In accordance with this suggestion, dopamine transporter
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(DAT) deletion results in a blockade of sensitization after repeated administration of amphetamine [28] or cocaine [29]. Furthermore, DAT-heterozygous mice show sensitization to amphetamine [28] but not to cocaine [29], a finding that underlines the importance of DAT in cocaine-induced sensitization processes. Psychostimulants are also able to alter other monoaminergic transporters. Thus, cocaine increases the vesicular monoamine transporter (VMAT2) activity [30]. The VMAT2 carries dopamine (and other monoamines) from the cytoplasm to storage vesicles from which they are released upon neural impulse and therefore is a major regulator of dopamine transmission. On testing the possible behavioral significance of this transporter system, Wang et al. [31] reported that VMAT2 heterozygous mice show supersensitivity to an acute administration of cocaine but they do not sensitize further. However, metamphetamine, which reduces VMAT2 activity [32], induced sensitization in VMAT2 heterozygous mice which shows that this transporter molecule does not play a unified role in psychostimulant-induced sensitization. Finally, studies in mice lacking the transporters for other monoamines have also produced inconclusive results. Regarding the norepinephrine transporter (NET), Xu et al. [33] observed that NET-knockout mice were supersensitive to the acute administration of cocaine or amphetamine and did not develop sensitization to cocaine (amphetamine was not evaluated). In contrast, Mead et al. [29] reported that NET-knockout mice do not respond to an acute cocaine challenge but they display a degree of sensitization comparable to that found in wild-type mice. The reason for this discrepancy is not clear, but procedural differences could be affecting the observed results (i.e. in the study of Mead et al. mice were extensively habituated in order to homogenize baseline locomomotion). Finally, although serotonin transporter (SERT), and the double DAT-SERT and DAT-NET knockout mice have also been developed, the possible consequences of these genotypes in the development/expression of psychostimulant sensitization has not yet been evaluated. Although the findings of the aforementioned studies in knockout mice have confirmed a role of the dopaminergic system in the development of psychostimulant-induced sensitization, in the last years it has become increasingly clear that other neurotransmitter systems also play a major role in this process [17]. However, the use of transgenic mice to corroborate the participation of these other neurotransmitter systems on psychostimulant sensitization has been rather scarce (for a brief overview see [1]). On the other hand, one of the major advances in using transgenic mice has been the possibility to modify the availability of some neural components that cannot be easily targeted by using a pharmacological approach (i.e. transcription factors). Thus, for example, the use of transgenic mice is definitively contributing to our understanding of the role of Fos family transcription factors in psychostimulant sensitization. In this regard, it has been shown that acute
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administration of psychostimulants induces the expression of several Fos-related elements, whereas the chronic administration of these drugs do not induce these proteins but instead causes the long-lasting expression and accumulation of highly stable forms of DfosB [34]. In agreement with these data, it has been shown that the deletion of FosB results in a potentiation to the locomotor changes elicited by an acute administration of cocaine but these animals do not show further enhancement of locomotion (sensitization) after the repeated administration of this substance [35]. However, transgenic mice overexpressing DfosB show an enhanced response to cocaine that persists after repeated injections [36]. Transgenic mice were also very helpful to elucidate the role of clock genes in psychostimulant-induced sensitization processes. Previous studies had established that Drosophila mutants of period ( per) and other clock genes (i.e. clock, cycle and double time) are required for normal response to acute cocaine as well as for the development to sensitization [37]. The availability of transgenic mice lacking the two mouse homolog period genes (mPer1 and mPer2) allowed the testing for this relationship in a mammal model. Thus, it was observed that although these animals are normal in the acute response to cocaine, mPer1- and mPer2-mutant mice show either an abolishment or enhancement in cocaine sensitization, respectively [38]. The mechanism underlying this interaction between circadian rhythmicity and cocaine sensitization is not completely understood, although it is becoming apparent that it may be related to the ability of these genes to regulate dopamine receptor gene expression [39,40]. In summary, from the studies reviewed above it became clear that the use of transgenic mice has produced two major advances in our understanding about psychostimulantinduced sensitization. First, they have been used in confirmatory studies to corroborate the implication of the dopaminergic system in the development/expression of this phenomenon. On the other hand the use of transgenic mice has allowed the study of the role of some molecular targets such as transcription factors, which are almost unreachable by other means.
4. Mouse models to study relapse behavior 4.1. Reinstatement of extinguished drug-seeking behavior under operant conditions in mice In the current issue the reinstatement model of drugseeking behavior in rats is described in detail by Weiss. We would like to add only some further information concerning the use of mice in the reinstatement procedure. Although the reinstatement model of drug-seeking behavior is a wellestablished model in rats [2], little effort has been made so far to transfer this model to mice. Reasons for this methodological transfer problem from rats to mice are
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obvious: some operant tasks have to be achieved by the individual during the course of a reinstatement experiment and usually rats acquire goal-directed behavior more easily than mice. The first goal which has to be achieved by an individual in the reinstatement procedure is selective responding on a reinforced lever. Usually this is an easy task for a rat; however, for mice, due to higher motor activity it is more difficult for them to achieve this task. Another confounding variable in mice is that lever pressing is reinforcing per se. The same problem might also relate to nose-poking. Thus whether a reinforcer follows a lever press/nose poke or not, it might not influence further behavior of the mouse. Despite the problems of high motor activity and reinforcing effects of lever pressing/nosepoking, mice usually acquire selective responding under a simple fixed ratio 1 (FR1) for the drug although on the control lever – the non-reinforced lever – a higher number of responses is seen than in rats. This statement, however, relates to a ‘weak reinforcer’ such as alcohol. If a drug, e.g. cocaine, produces a stronger stimulus control, selective responding occurs faster and is more selective, which means that similar to rats, low spontaneous responding occurs on the non-reinforced lever. The next task which has to be achieved is extinction of lever responding. Thus lever responding is without any further consequence and the individual does not receive the drug any longer. Rats usually show a short burst of responding and then responding gradually declines over a period of days. Usually following 10 days rats show only some spontaneous responses; however, this occurs at a low rate. Extinction in mice seems to be more difficult and one needs more extinction days as compared to rats, and again much higher rates of spontaneous responding is seen. In the third task reinstatement of drug-seeking behavior has to be elicited. So far only two systematic studies in mice on the reinstatement paradigm have been published. The group of Shaham recently showed in 129X1/SvJ mice – the most common strain for embryonic stem cell manipulation – that cocaine cues and food deprivation can reinstate extinguished responding for cocaine, however, cocaine priming produced modest effects on reinstatement [41]. A similar systematic work was performed in C57BL/6J mice – the most common strain for behavioral studies (most transgenic mice are usually backcrossed to this particular strain) – showing again robust reinstatement following the presentation of conditioned cues; however, cocaine priming even up to a 40 mg/kg dose was without any consequence in this strain [42]. These findings somehow mirror the situation in rats in the cocaine reinstatement procedure. Thus conditioned cues serve as powerful triggers to reinstate cocaineseeking behavior whereas cocaine priming produces, if at all, only modest reinstatement. In summary, both studies demonstrate that the reinstatement paradigm can in fact be transferred from the rat to the mouse and that this sophisticated model can be now used to study transgenic mice.
4.2. Reinstatement of extinguished drug-seeking behavior in a conditioned place preference paradigm in mice In the context of reinstatement behavior, it should also be mentioned that some efforts have been made to study reinstatement of drug-induced conditioned place preference in mice. In a typical experiment mice are daily injected with the drug and paired with a specific compartment. On alternating days, the animals receive saline injections and are then paired with a distinguishable compartment in a conditioning box. After several days of conditioning, a drug-induced conditioned place preference is achieved which is then extinguished with repeated saline injections in both the previously drug-paired compartment and the saline-paired compartment. Following the extinction phase, the reinstatement of conditioned place preference is initiated by drug priming. Thus the priming injection of the drug induces a marked preference for the previously drug-paired compartment. This procedure has already been used in different outbred mouse strains and it has been demonstrated that cocaine [43], as well as ethanol priming [44], can reinstate extinguished drug-seeking behavior in a conditioned place preference paradigm. Although this procedure is straightforward and mouse transgenics can easily be studied in this paradigm, it seems that more systematic work is still required in order to fully understand the conceptual background of this paradigm. Thus, in a recent study it has been shown that conditioned as well as unconditioned factors contribute to the reinstatement of cocaine place conditioning in C57BL/6J mice [45].
4.3. Second order schedules in mice On a second-order schedule, an individual’s responding is maintained not only by the self-administered drug, but also by contingent presentation of drug-paired stimuli that serve as conditioned reinforcers of instrumental behavior (for recent reviews see [46,47]). One of the advantages of second-order schedules of drug injection is that they maintain high rates of responding and that they therefore allow the study of more complex behavioral sequences than do simple schedules. Much of the early work in this area used primates as subjects and focused on the behavioral variables controlling responding. The recent extension of second-order self-administration studies to rats as subjects has facilitated the investigation of neural mechanisms involved in this behavior. Even though this paradigm has been used extensively in the last decade and reflects several aspects of the human drug abuse situation, we are aware of only one conditioned reward study in mice that used a second-order schedule [48]. Clearly more effort has to be invested to extend this paradigm finally to transgenic mouse work.
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4.4. The alcohol deprivation effect in mice Another model which is frequently used in alcohol research is the alcohol deprivation effect model. After several weeks of voluntary alcohol consumption, the drugtaking behavior following a deprivation phase is characterized by a transient phase of increased alcohol intake and preference. During this so-called alcohol deprivation effect (ADE; i.e. relapse-like drinking behavior), rats exhibit a high motivation for alcohol and show a shift in ethanol preference towards highly concentrated alcohol solutions [49,50]. This robust phenomenon is observed across several species including monkeys and human social drinkers, and Salimov and Salimova [51] were the first who described this effect in mice. In this study, and also in subsequent studies of the same group [51,52] many mice failed to exhibit an alcohol deprivation effect; however, it is important to note that only a short deprivation period of 3 days has been introduced in these studies which might lead to a lack of effect in most of the mice. The experiences in our laboratory over the past years have shown that a deprivation period of at least 2 –3 weeks leads to an alcohol deprivation effect in most mice. However, there are always a number of mice which consistently fail to show such an effect. We are now in the course of a selective breeding program where we breed two lines of mice which show a consistent alcohol deprivation effect versus mice which consistently fail to show such an effect. These two lines will certainly help to identify the genetic factors underlying the alcohol deprivation effect and thereby alcohol relapse drinking behavior. 4.5. The use of transgenic mice in relapse models As described, presently drug abuse researchers are in the course of adapting relapse models to mice, and therefore it is no surprise that only very few relapse studies have been conducted so far in transgenic mice. Using the reinstatement paradigm we are aware of one preliminary report with mice carrying a mutation in genes that control our central clock, namely the per gene 1 and 2 (mPer1 and mPer2) [53]. In this study, the animals were trained to self-administer ethanol (10%) using a standard sucrose fading procedure under an FR1 paradigm. Ethanol reinforcement was paired with a light stimulus. Following the establishment of a stable baseline for ethanol responding all animals underwent extinction, e.g. responses on the ethanol lever were not reinforced any longer. Following 20 days of extinction, responding stabilized on a relatively low level and ethanolseeking behavior could be elicited by the conditioned stimuli. However, the combination of the conditioned stimuli and ethanol presentation led to a more pronounced reinstatement behavior in all three lines of mice. A specific genotype effect could not be detected for reinstatement behavior; however, the mPer2 transgenic mice showed in general higher responding for ethanol and therefore had higher ethanol intake compared to wild-type animals [53].
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These results suggest a relationship between the circadian clock gene mPer2 and ethanol reinforcement; however, relapse behavior seems not to be affected by this mutation, which is in line with our observation in the alcohol deprivation effect model: Although mPer2 transgenic mice drink much more alcohol under free-choice conditions, they do not differ in the expression of the alcohol deprivation effect from wild-type mice [40]. To our knowledge, reinstatement of extinguished drugseeking behavior in a conditioned place preference paradigm in transgenic mice has not been used so far; although, this is the most simple model to study the effects of gene manipulation in mice on relapse behavior. There is a recent application; however, of a second-order schedule in AMPA receptor subunit (GluR1) deficient mice [48]. These mice displayed impaired acquisition of responding under a second-order schedule, suggesting a specific deficit in conditioned reward. In this study the authors suggest that specific antagonists that block GluR1 containing AMPA receptors may offer a new approach to disrupting drug-seeking behavior that is maintained by cues conditioned to drug taking, without affecting other aspects of learning and memory. However, this conclusion is limited by a recent finding in GluR1 knockout animals which shows that these animals display a normal alcohol deprivation effect [54], a finding which leads us to the conclusion that the GluR1 subunit of the AMPA receptor does not seem to play a critical role in the etiology of alcohol dependence and relapse behavior. Although the GluR1 subunit of the AMPA receptor might not be directly involved in relapse behavior to alcohol, other AMPA subunits or other glutamate receptors might play a more prominent role in alcohol relapse since it is suggested that a hyperglutamatergic system is mainly triggering this behavior [55]. Since the phosphorylation status of glutamate receptors is modulated by Fyn-tyrosine kinase, we were interested whether Fyn deficiency in knockout mice would lead to an altered relapse-like drinking behavior as measured by the alcohol deprivation effect. However, no genotype effect could be detected in this model, suggesting that phosphorylation of glutamate receptor through this particular kinase is not involved in alcohol relapse [56]. In summary, only a few reports on transgenic mice in relapse models are available and the information from these studies has had, so far, no great influence on better treatment strategies for relapse behavior. However, hopefully it became clear to the reader that we are just entering a new research area and we strongly believe that in the future many laboratories working on relapse will follow this line of research, and will produce helpful information to guide us in new treatment strategies on relapse behavior.
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[50] McBride WJ, Le AD, Noronha A. Central nervous system mechanisms in alcohol relapse. Alcohol Clin Exp Res 2002;26: 280–6. [51] Salimov RM, Salimova NB. The alcohol-deprivation effect in hybrid mice. Drug Alcohol Depend 1993;32:187–91. [52] Salimov RM, Salimova NB, Shvets LN, Maisky AI. Haloperidol administered subchronically reduces the alcohol-deprivation effect in mice. Alcohol 2000;20:61–8. [53] Zghoul T, Sanchis C, Abarca C, Albrecht U, Spanagel R. Operant ethanol self-administration and reinstatement behavior in mPer1 and mPer2 knockout mice. Behav Pharmacol 2003;14(Suppl 1):S67. [54] Cowen MS, Schroff KC, Sprengel R, Spanagel R. Neurobehavioral effects of alcohol in AMPA receptor subunit (GluR1) deficient mice. Neuropharmacology 2003;45:325–33. [55] Spanagel R, Bienkowski P. Glutamatergic mechanisms in alcohol dependence and addiction. In: Lodge D, Danysz W, Parsons CG, editors. Therapeutic potential of ionotropic glutamate receptor antagonists and modulators. Johnson City, TN: Graham; 2002. p. 375 –403. [56] Cowen M, Schumann G, Yagi T, Spanagel R. Role of Fyn tyrosine kinase in ethanol consumption by mice. Alcohol Clin Exp Res 2003; 27:1213–9.
The use of transgenic mice to study addictive behavior Rainer Spanagela,*, Carles Sanchis-Seguraa,b a
Department of Psychopharmacology, Central Institute of Mental Health, J 5, D-68159 Mannheim, Germany b Departament de Psicologia Ba`sica, Clı´nica i Psicobiologia, Universitat Jaume I, Castello, Spain
Abstract Drug addiction is a behavioral disorder which arises in some individuals after repeated drug use. In these individuals, the search and consumption of the drug becomes compulsive and takes place at expense of all other vital activities, resulting in a complete loss of social compatibility (e.g. loss of partner and friends, loss of job, crime). From this definition two main consequences can be obtained. First, addiction is not a direct and inevitable consequence of drug use, a fact that suggests the existence of identifiable factors that can contribute to the vulnerability to addiction. Second, it is also apparent that addiction cannot be fully modeled in preclinical animal models. However, some specific features of addiction such as the existence of relapses even after a long period of abstinence can be successfully studied in different animal models. Moreover, different tests which do not directly reproduce any behavioral features of addictive behavior (e.g. locomotor sensitization) have been proved to have a high heuristic value in unraveling the neurobiological changes associated in the transition that leads from a casual use of a drug to the addictive state. In the present review we summarize the studies devoted to identify genetic factors that could result in a higher susceptibility to addiction by using genetically engineered mice in the preclinical model and tests available. From this overview it is concluded that, compared to the number of studies about drug self-administration, the use of transgenic mice in addictionrelated models has been rather scarce and should be further encouraged. q 2003 Elsevier B.V. All rights reserved. Keywords: Mouse; Knockout; Addiction; Sensitization; Relapse
1. Introduction Drug addiction is defined as a syndrome in which drug use (e.g. psychostimulants, opiates, alcohol) pervades all life activities of the user. The life becomes governed by the drug and the addicted patient can lose social compatibility (e.g. loss of partner and friends, loss of job, crime). Behavioral characteristics of this syndrome are compulsive drug use, craving and chronic relapses which can occur even after years of abstinence. How can addictive behavior be measured in laboratory animals? Some years ago we had an expert meeting on animal models of addiction. In the course of the discussion, it became clear that terms such as ‘compulsive drug use’ or ‘craving’ are difficult to transfer to laboratory animals and that only some aspects of addictive behavior can be studied in laboratory animals. However, one phenomenon which might give new insights into drug craving and can easily be studied in mice is drug-induced sensitization. Therefore, numerous studies using transgenic mice have been * Corresponding author. Tel.: þ49-621-170-3833; fax: þ 49-621-1703837. E-mail address: [email protected] (R. Spanagel). 1566-2772/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1566-2772(03)00094-X
published on drug-induced sensitization in the past decade [1]. Here we will present an overview of these studies. Other aspects of addictive behavior, such as relapse behavior, can be studied more directly in laboratory animals. The standard procedure used in most laboratories working on relapse behavior is the operant reinstatement model of drug-seeking behavior [2]. This model can be used to study relapse to all kind of drugs. However, other models have also been used to study relapse behavior in laboratory animals. In the second part of this review we will briefly describe these models on relapse behavior and their special application to mice. Then we will discuss studies using these procedures to examine transgenic mice for the elucidation of genes involved in relapse behavior.
2. Drug-induced sensitization in mice The term sensitization refers to an increase in a response after the repeated occurrence of the stimuli that promotes it. Sensitization in drug abuse research has been mainly studied in respect to locomotor activity. Thus the ability of addictive drugs to increase locomotion, after an acute administration,
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is progressively enhanced when drug exposure is repeated. This so-called psychomotor sensitization is a very robust phenomenon that has been observed across several species including mice. Although there are variations among sensitization protocols for mice, to achieve this effect, any sensitization protocol should use a route of administration with a fast onset of drug effect (usually intraperitoneal or intravenous injections are used) and the drug has to be given intermittently. Moreover, sensitization is stronger when the dose is higher or when escalating doses are administered [3,4]. Some drugs such as psychostimulants [5] and morphine [6] can even trigger a sensitized response after a single pre-exposure if the dose used was high enough. Finally, although sensitization is in essence a nonassociative process and it can be triggered independently of any environmental cue, the context can play a major role in the development and expression of sensitization [7,8]. Thus a higher degree of sensitization is usually observed when drug injections are administered in a context different from the home cage and subjects fail to express sensitization when they are challenged in a different context where they have never experienced the drug [9,10]. It has further been proposed that the incentive salience attributed to an initially neutral stimulus is progressively increased when this stimuli is contingent and repeatedly associated to the drug administration, therefore leading to an ‘incentive sensitization’ [8]. In this regard, the sensitization process has become one of the most commonly proposed mechanisms to explain the transition from a regular pattern of voluntary drug intake to compulsive drug-seeking and taking behavior [8,11]. However, to test the occurrence of this phenomenon a very sophisticated experimental approach is required in order to rule out other confounding processes (i.e. changes in the hedonic value of the drug). For this reason, no standard protocols to test incentive sensitization are currently available and those used till now have been only applied to rats [12,13] since the acquisition of complex contingencies related to these tests cannot be easily learned by mice. Therefore, although it is clear that the incentive sensitization may provide a more direct link to the core features of addictive behavior, psychomotor sensitization in mice has been used far more in the past.
3. The use of transgenic mice in drug-induced sensitization studies It is suggested that psychomotor and incentive sensitization are at least partially mediated by the same neural circuitry, i.e. nucleus accumbens; therefore, both phenomena are useful behavioral manifestations to assess neuroplastic changes of this neural system in relationship to drug-seeking behavior. Most of the studies about druginduced sensitization are related to repeated exposure to
psychostimulants, since these drugs act primarily on dopaminergic neurons and produce a more robust sensitization than other addictive drugs. Therefore, in this paragraph we will focus on psychostimulant-induced sensitization. It has been demonstrated that psychostimulants potentiate dopamine signaling in the mesocorticolimbic system and this effect is progressively enhanced as the administration of these drugs is repeated [14 – 16]. Thus, it has been suggested that transient increases in somatodendritic dopamine release and spontaneous firing activity of dopamine neurons in the ventral tegmental area (VTA) could be related with the induction of behavioral sensitization, whereas long-lasting alterations within dopamine neuronal terminal fields could be responsible for its expression [17]. The exact nature of these changes is not completely understood, but changes in several neural substrates involved in dopamine neurotransmission have been described, and in the last years transgenic mice have been used to confirm the involvement of these molecular components in the development/expression of psychostimulant-induced sensitization. Among these changes, the role of dopamine receptors in amphetamine or cocaine-induced sensitization has been extensively studied [18]. Thus, it has been demonstrated that D1-dopamine receptor knockout mice display a reduction in their locomotion after an acute injection of cocaine and also an attenuated sensitization response to cocaine [19]. Surprisingly, these mice show normal sensitization to amphetamine [20,21]. On the other hand, after repeated psychostimulant administration there is reduction in the sensitivity of D2-dopamine autoreceptors that could contribute to enhanced dopamine neurotransmission in the nucleus accumbens [17]. However, although this effect could play a role in the development of sensitization, the contribution of this effect to the expression of sensitization seems very unlikely because alterations of D2-dopamine autoreceptors are transient in nature compared to the persistence of the behavioral manifestation of drug-induced sensitization. In agreement with this suggestion, it has been demonstrated that the D2-dopamine receptor knockout mice display normal locomotion and sensitization after acute and chronic cocaine administration respectively [22]. Conversely, D3-dopamine receptor knockout mice display an enhanced response to an acute challenge with cocaine [23] but exhibit a normal sensitized response after repeated injections of this drug [24]. Finally, the D4-dopamine receptor knockout mice show an enhanced response to an acute challenge with cocaine or metamphetamine [25,26] but their response to repeated injections of these drugs have not yet been evaluated. It is also suggested that alterations in monoamine transporter systems – which are the primary site of action of psychostimulants – are involved in the development and expression of psychostimulant-induced sensitization [27]. In accordance with this suggestion, dopamine transporter
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(DAT) deletion results in a blockade of sensitization after repeated administration of amphetamine [28] or cocaine [29]. Furthermore, DAT-heterozygous mice show sensitization to amphetamine [28] but not to cocaine [29], a finding that underlines the importance of DAT in cocaine-induced sensitization processes. Psychostimulants are also able to alter other monoaminergic transporters. Thus, cocaine increases the vesicular monoamine transporter (VMAT2) activity [30]. The VMAT2 carries dopamine (and other monoamines) from the cytoplasm to storage vesicles from which they are released upon neural impulse and therefore is a major regulator of dopamine transmission. On testing the possible behavioral significance of this transporter system, Wang et al. [31] reported that VMAT2 heterozygous mice show supersensitivity to an acute administration of cocaine but they do not sensitize further. However, metamphetamine, which reduces VMAT2 activity [32], induced sensitization in VMAT2 heterozygous mice which shows that this transporter molecule does not play a unified role in psychostimulant-induced sensitization. Finally, studies in mice lacking the transporters for other monoamines have also produced inconclusive results. Regarding the norepinephrine transporter (NET), Xu et al. [33] observed that NET-knockout mice were supersensitive to the acute administration of cocaine or amphetamine and did not develop sensitization to cocaine (amphetamine was not evaluated). In contrast, Mead et al. [29] reported that NET-knockout mice do not respond to an acute cocaine challenge but they display a degree of sensitization comparable to that found in wild-type mice. The reason for this discrepancy is not clear, but procedural differences could be affecting the observed results (i.e. in the study of Mead et al. mice were extensively habituated in order to homogenize baseline locomomotion). Finally, although serotonin transporter (SERT), and the double DAT-SERT and DAT-NET knockout mice have also been developed, the possible consequences of these genotypes in the development/expression of psychostimulant sensitization has not yet been evaluated. Although the findings of the aforementioned studies in knockout mice have confirmed a role of the dopaminergic system in the development of psychostimulant-induced sensitization, in the last years it has become increasingly clear that other neurotransmitter systems also play a major role in this process [17]. However, the use of transgenic mice to corroborate the participation of these other neurotransmitter systems on psychostimulant sensitization has been rather scarce (for a brief overview see [1]). On the other hand, one of the major advances in using transgenic mice has been the possibility to modify the availability of some neural components that cannot be easily targeted by using a pharmacological approach (i.e. transcription factors). Thus, for example, the use of transgenic mice is definitively contributing to our understanding of the role of Fos family transcription factors in psychostimulant sensitization. In this regard, it has been shown that acute
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administration of psychostimulants induces the expression of several Fos-related elements, whereas the chronic administration of these drugs do not induce these proteins but instead causes the long-lasting expression and accumulation of highly stable forms of DfosB [34]. In agreement with these data, it has been shown that the deletion of FosB results in a potentiation to the locomotor changes elicited by an acute administration of cocaine but these animals do not show further enhancement of locomotion (sensitization) after the repeated administration of this substance [35]. However, transgenic mice overexpressing DfosB show an enhanced response to cocaine that persists after repeated injections [36]. Transgenic mice were also very helpful to elucidate the role of clock genes in psychostimulant-induced sensitization processes. Previous studies had established that Drosophila mutants of period ( per) and other clock genes (i.e. clock, cycle and double time) are required for normal response to acute cocaine as well as for the development to sensitization [37]. The availability of transgenic mice lacking the two mouse homolog period genes (mPer1 and mPer2) allowed the testing for this relationship in a mammal model. Thus, it was observed that although these animals are normal in the acute response to cocaine, mPer1- and mPer2-mutant mice show either an abolishment or enhancement in cocaine sensitization, respectively [38]. The mechanism underlying this interaction between circadian rhythmicity and cocaine sensitization is not completely understood, although it is becoming apparent that it may be related to the ability of these genes to regulate dopamine receptor gene expression [39,40]. In summary, from the studies reviewed above it became clear that the use of transgenic mice has produced two major advances in our understanding about psychostimulantinduced sensitization. First, they have been used in confirmatory studies to corroborate the implication of the dopaminergic system in the development/expression of this phenomenon. On the other hand the use of transgenic mice has allowed the study of the role of some molecular targets such as transcription factors, which are almost unreachable by other means.
4. Mouse models to study relapse behavior 4.1. Reinstatement of extinguished drug-seeking behavior under operant conditions in mice In the current issue the reinstatement model of drugseeking behavior in rats is described in detail by Weiss. We would like to add only some further information concerning the use of mice in the reinstatement procedure. Although the reinstatement model of drug-seeking behavior is a wellestablished model in rats [2], little effort has been made so far to transfer this model to mice. Reasons for this methodological transfer problem from rats to mice are
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obvious: some operant tasks have to be achieved by the individual during the course of a reinstatement experiment and usually rats acquire goal-directed behavior more easily than mice. The first goal which has to be achieved by an individual in the reinstatement procedure is selective responding on a reinforced lever. Usually this is an easy task for a rat; however, for mice, due to higher motor activity it is more difficult for them to achieve this task. Another confounding variable in mice is that lever pressing is reinforcing per se. The same problem might also relate to nose-poking. Thus whether a reinforcer follows a lever press/nose poke or not, it might not influence further behavior of the mouse. Despite the problems of high motor activity and reinforcing effects of lever pressing/nosepoking, mice usually acquire selective responding under a simple fixed ratio 1 (FR1) for the drug although on the control lever – the non-reinforced lever – a higher number of responses is seen than in rats. This statement, however, relates to a ‘weak reinforcer’ such as alcohol. If a drug, e.g. cocaine, produces a stronger stimulus control, selective responding occurs faster and is more selective, which means that similar to rats, low spontaneous responding occurs on the non-reinforced lever. The next task which has to be achieved is extinction of lever responding. Thus lever responding is without any further consequence and the individual does not receive the drug any longer. Rats usually show a short burst of responding and then responding gradually declines over a period of days. Usually following 10 days rats show only some spontaneous responses; however, this occurs at a low rate. Extinction in mice seems to be more difficult and one needs more extinction days as compared to rats, and again much higher rates of spontaneous responding is seen. In the third task reinstatement of drug-seeking behavior has to be elicited. So far only two systematic studies in mice on the reinstatement paradigm have been published. The group of Shaham recently showed in 129X1/SvJ mice – the most common strain for embryonic stem cell manipulation – that cocaine cues and food deprivation can reinstate extinguished responding for cocaine, however, cocaine priming produced modest effects on reinstatement [41]. A similar systematic work was performed in C57BL/6J mice – the most common strain for behavioral studies (most transgenic mice are usually backcrossed to this particular strain) – showing again robust reinstatement following the presentation of conditioned cues; however, cocaine priming even up to a 40 mg/kg dose was without any consequence in this strain [42]. These findings somehow mirror the situation in rats in the cocaine reinstatement procedure. Thus conditioned cues serve as powerful triggers to reinstate cocaineseeking behavior whereas cocaine priming produces, if at all, only modest reinstatement. In summary, both studies demonstrate that the reinstatement paradigm can in fact be transferred from the rat to the mouse and that this sophisticated model can be now used to study transgenic mice.
4.2. Reinstatement of extinguished drug-seeking behavior in a conditioned place preference paradigm in mice In the context of reinstatement behavior, it should also be mentioned that some efforts have been made to study reinstatement of drug-induced conditioned place preference in mice. In a typical experiment mice are daily injected with the drug and paired with a specific compartment. On alternating days, the animals receive saline injections and are then paired with a distinguishable compartment in a conditioning box. After several days of conditioning, a drug-induced conditioned place preference is achieved which is then extinguished with repeated saline injections in both the previously drug-paired compartment and the saline-paired compartment. Following the extinction phase, the reinstatement of conditioned place preference is initiated by drug priming. Thus the priming injection of the drug induces a marked preference for the previously drug-paired compartment. This procedure has already been used in different outbred mouse strains and it has been demonstrated that cocaine [43], as well as ethanol priming [44], can reinstate extinguished drug-seeking behavior in a conditioned place preference paradigm. Although this procedure is straightforward and mouse transgenics can easily be studied in this paradigm, it seems that more systematic work is still required in order to fully understand the conceptual background of this paradigm. Thus, in a recent study it has been shown that conditioned as well as unconditioned factors contribute to the reinstatement of cocaine place conditioning in C57BL/6J mice [45].
4.3. Second order schedules in mice On a second-order schedule, an individual’s responding is maintained not only by the self-administered drug, but also by contingent presentation of drug-paired stimuli that serve as conditioned reinforcers of instrumental behavior (for recent reviews see [46,47]). One of the advantages of second-order schedules of drug injection is that they maintain high rates of responding and that they therefore allow the study of more complex behavioral sequences than do simple schedules. Much of the early work in this area used primates as subjects and focused on the behavioral variables controlling responding. The recent extension of second-order self-administration studies to rats as subjects has facilitated the investigation of neural mechanisms involved in this behavior. Even though this paradigm has been used extensively in the last decade and reflects several aspects of the human drug abuse situation, we are aware of only one conditioned reward study in mice that used a second-order schedule [48]. Clearly more effort has to be invested to extend this paradigm finally to transgenic mouse work.
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4.4. The alcohol deprivation effect in mice Another model which is frequently used in alcohol research is the alcohol deprivation effect model. After several weeks of voluntary alcohol consumption, the drugtaking behavior following a deprivation phase is characterized by a transient phase of increased alcohol intake and preference. During this so-called alcohol deprivation effect (ADE; i.e. relapse-like drinking behavior), rats exhibit a high motivation for alcohol and show a shift in ethanol preference towards highly concentrated alcohol solutions [49,50]. This robust phenomenon is observed across several species including monkeys and human social drinkers, and Salimov and Salimova [51] were the first who described this effect in mice. In this study, and also in subsequent studies of the same group [51,52] many mice failed to exhibit an alcohol deprivation effect; however, it is important to note that only a short deprivation period of 3 days has been introduced in these studies which might lead to a lack of effect in most of the mice. The experiences in our laboratory over the past years have shown that a deprivation period of at least 2 –3 weeks leads to an alcohol deprivation effect in most mice. However, there are always a number of mice which consistently fail to show such an effect. We are now in the course of a selective breeding program where we breed two lines of mice which show a consistent alcohol deprivation effect versus mice which consistently fail to show such an effect. These two lines will certainly help to identify the genetic factors underlying the alcohol deprivation effect and thereby alcohol relapse drinking behavior. 4.5. The use of transgenic mice in relapse models As described, presently drug abuse researchers are in the course of adapting relapse models to mice, and therefore it is no surprise that only very few relapse studies have been conducted so far in transgenic mice. Using the reinstatement paradigm we are aware of one preliminary report with mice carrying a mutation in genes that control our central clock, namely the per gene 1 and 2 (mPer1 and mPer2) [53]. In this study, the animals were trained to self-administer ethanol (10%) using a standard sucrose fading procedure under an FR1 paradigm. Ethanol reinforcement was paired with a light stimulus. Following the establishment of a stable baseline for ethanol responding all animals underwent extinction, e.g. responses on the ethanol lever were not reinforced any longer. Following 20 days of extinction, responding stabilized on a relatively low level and ethanolseeking behavior could be elicited by the conditioned stimuli. However, the combination of the conditioned stimuli and ethanol presentation led to a more pronounced reinstatement behavior in all three lines of mice. A specific genotype effect could not be detected for reinstatement behavior; however, the mPer2 transgenic mice showed in general higher responding for ethanol and therefore had higher ethanol intake compared to wild-type animals [53].
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These results suggest a relationship between the circadian clock gene mPer2 and ethanol reinforcement; however, relapse behavior seems not to be affected by this mutation, which is in line with our observation in the alcohol deprivation effect model: Although mPer2 transgenic mice drink much more alcohol under free-choice conditions, they do not differ in the expression of the alcohol deprivation effect from wild-type mice [40]. To our knowledge, reinstatement of extinguished drugseeking behavior in a conditioned place preference paradigm in transgenic mice has not been used so far; although, this is the most simple model to study the effects of gene manipulation in mice on relapse behavior. There is a recent application; however, of a second-order schedule in AMPA receptor subunit (GluR1) deficient mice [48]. These mice displayed impaired acquisition of responding under a second-order schedule, suggesting a specific deficit in conditioned reward. In this study the authors suggest that specific antagonists that block GluR1 containing AMPA receptors may offer a new approach to disrupting drug-seeking behavior that is maintained by cues conditioned to drug taking, without affecting other aspects of learning and memory. However, this conclusion is limited by a recent finding in GluR1 knockout animals which shows that these animals display a normal alcohol deprivation effect [54], a finding which leads us to the conclusion that the GluR1 subunit of the AMPA receptor does not seem to play a critical role in the etiology of alcohol dependence and relapse behavior. Although the GluR1 subunit of the AMPA receptor might not be directly involved in relapse behavior to alcohol, other AMPA subunits or other glutamate receptors might play a more prominent role in alcohol relapse since it is suggested that a hyperglutamatergic system is mainly triggering this behavior [55]. Since the phosphorylation status of glutamate receptors is modulated by Fyn-tyrosine kinase, we were interested whether Fyn deficiency in knockout mice would lead to an altered relapse-like drinking behavior as measured by the alcohol deprivation effect. However, no genotype effect could be detected in this model, suggesting that phosphorylation of glutamate receptor through this particular kinase is not involved in alcohol relapse [56]. In summary, only a few reports on transgenic mice in relapse models are available and the information from these studies has had, so far, no great influence on better treatment strategies for relapse behavior. However, hopefully it became clear to the reader that we are just entering a new research area and we strongly believe that in the future many laboratories working on relapse will follow this line of research, and will produce helpful information to guide us in new treatment strategies on relapse behavior.
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