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Neuron, Vol. 21, 467-476, September, 1998, Copyright 01998 by Cell Press Neuroscience of Addiction George F. Koob,’ Pietro Paolo Sanna, and Floyd E. Bloom Department of Neuropharmacology The Scripps Research Institute La Jolla, California 92037 Human addictions are chronically relapsing disorders characterized by compulsive drug taking, an inability to limit the intake of drugs, and the emergence of a withdrawal syndrome during cessation of drug taking (dependence). The development of an addiction impacts on several separate neurobiological processes, and these effects are both drug- and drug use-dependent. In animal models of addiction, changes in specific neurotransmitter systems within a highly limited band of structures, including specific parts of the nucleus accumbens and amygdala, may underlie drug reward and the motivational effects associated with dependence. Changes in the signals mediated by several neurotransmitters, including dopamine, opioid peptides, and corticotropin-releasing factor (CRF), and in the regulation of selected transcription factors within the neurons of this reward circuit, may underlie the vulnerability to relapse that characterizes addiction in humans. Animal Models of Addiction Addiction, also known as substance dependence (American Psychiatric Association, 1994), is a chronically relapsing disorder that is characterized by three major elements: (1) compulsion to seek and take the drug, (2) loss of control in limiting intake, and (3) emergence of a negative emotional state (e.g., dysphoria, anxiety, irritability) when access to the drug is prevented (defined here as dependence) (Koob and Le Moal, 1997). Both clinically and in experimental animals, the occasional use of an abusable drug is distinct from repeated drug use and the emergence of chronic drug addiction. The goal of current neuroscience research is to understand the cellular and molecular mechanisms that mediate the transition between occasional, controlled drug use and the loss of behavioral control over drug seeking and drug taking that defines chronic addiction (Koob and Le Moal, 1997). Much of the recent progress in understanding the mechanisms of addiction has derived from the development of animal models of addiction on specific drugs such as opiates, stimulants, and ethanol. These animal models have localized the synaptic sites and transductive mechanisms in the nervous system on which drugs of abuse act initially and are beginning to be used to explore how the nervous system adapts to drug use. While no animal model of addiction fully emulates the human condition, animal models do permit investigation of elements of the process of drug addiction, and the data derived from these models provides an empirical ‘To whom correspondence should be addressed. Review framework for understanding the molecular basis of addiction (Koob and Le Moal, 1997; Koob et al., 1998). The underlying molecular and cellular changes that occur with the transition from occasional drug use to pathological abuse and addiction are only partially understood as yet. To reach a more complete understanding, these events will have to be integrated with the animal models of the different elements of the addiction process, including models of the transition from simple drug taking to compulsive use at the molecular, cellular, and behavioral levels. In this review, we will focus in particular on the factors that drive drug-seeking behavior at different stages of the addiction cycle (Koob and Le Moal, 1997), and we will place particular emphasis on trying to identify what is currently known and what remains to be elucidated. Neurobiological Substrates for the Acute Reinforcing Effects of Drugs of Abuse Animals and humans will readily self-administer the same classes of drugs, and such self-administration defines these drugs as positive reinforcers (Headlee et al., 1955). The powerful reinforcing properties of such drugs are revealed by the efforts experimental animals will perform to get them, such as pressing a lever multiple times to receive an intravenous injection of the drug. While early work focused on drug-taking behaviors in dependent animals (largely primates), subsequent studies have replicated the same behaviors in nondependent animals (largely rodents) (Schuster and Thompson, 1969). Since rodents are more tractable experimentally, they have provided much recent insight into the pertinent circuits and transductive mechanisms for the acute reinforcing effects of drugs of abuse (Koob and Bloom, 1988). Neuropharmacological studies have established an important role for the dopaminergic system in the acute reinforcing effects of cocaine (see Table 1) (Woolverton and Johnson, 1992). The midbrain dopamine system is composed of two major projections: the nigrostriatal system, which projects from the substantia nigra to the corpus striatum, and the mesocorticolimbic dopamine system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens, olfactory tubercle, frontal cortex, and amygdala. It is the mesocorticolimbic system that has been primarily implicated in the reinforcing actions of drugs of abuse. Psychostimulants such as cocaine and d-amphetamine elevate extracellular dopamine by inhibiting reuptake of dopamine by the dopamine transporter and, in the case of d-amphetamine, also by promoting reverse transport of dopamine. During intravenous cocaine self-administration, increased extracellular dopamine can be detected in the nucleus accumbens using in vivo microdialysis (Pettit and Justice, 1989). In addition, selective destruction of mesocorticolimbic dopamine neurons with the neurotoxin 6-hydroxydopamine (6-OHDA) eliminates cocaine selfadministration; similar decreases in cocaine self-administration occur when 6-OHDA lesions are restricted to . s Neuron 468 Table 1. Neurobiological Substrates for the Acute Reinforcing Effects of Drugs of Abuse Drug of Abuse Neurotransmitter Sites Cocaine and amphetamines Opiates Dopamine Serotonin Dopamine Opioid peptides Dopamine Opioid peptides? Nucleus accumbens Amygdala Ventral tegmental area Nucleus accumbens Ventral tegmental area Nucleus accum bens Amygdala? Ventral tegmental area Nicotine THC Ethanol Dopamine Opioid peptides? Dopamine Opioid peptides Serotonin GABA Glutamate Ventral tegmental area Nucleus accumbens Amygdala the dopamine fibers of the region of the nucleus accumbens (Roberts et al., 1980). Multiple receptor subtypes exist for transducing the increase in extracellular dopamine induced by psychomotor stimulants into behavioral action. Antagonists for the dopamine Dl, 02, and 03 receptor subtypes all decrease the reinforcing properties of cocaine (Woolverton and Johnson, 1992; Caine et al., 1995; Koob and Le Moal, 1997; Epping-Jordan et al., 1998a). The neuronal interaction responsible for cocaine reinforcement and the motivation to seek the drug appears to reside within the nucleus accumbens (Chang et al., 1994; Carelli and Deadwyler, 1996; Peoples et al., 1997). Electrophysiological recordings in animals receiving intravenous cocaine by self-administration have identified several patterns of neuronal responses in the nucleus accumbens, all time-locked to the self-administered drug infusion. One group of neurons fires just before the lever press, and this anticipatory response may be an initiation or trigger mechanism. A second group of neurons appears to change firing rate only after the cocaine infusion, and these neurons may represent the direct effects of reinforcement (Carelli and Deadwyler, 1996). Other neurons fire in proportion to the interinfusion interval between consecutive self-administration responses (Peoples and West, 1996). However, there appears to be a fourth type of neuronal firing pattern unique to cocaine self-administration; these “cocainespecific cells” fire both before and after the cocainereinforced response (Carelli and Deadwyler, 1996). Even more intriguing is the observation that this subset of neurons also fires to sensory stimuli (sounds or lights) that have been experimentally paired with cocaine delivery. Nucleus accumbens neurons may therefore mediate conditioned drug responses (Carelli and Deadwyler, 1996). Similarly, conditioned sensory stimuli are strong elicitors of “craving” in cocaine-taking humans. Recent studies using recombinant DNA techniques to “knock out” specific genes involved in dopaminergic neurotransmission may provide evidence of some redundancy in the neurochemical basis of cocaine reinforcement. In a mouse strain in which the gene for the dopamine transporter was disrupted by homologous recombination, psychostimulants failed to alter baseline extracellular dopamine levels and failed to induce behavioral effects such as enhanced locomotor activity and stereotypy (Giros et al., 1996). However, such dopamine transporter-deficient mice still could be trained to self-administer cocaine despite persistently high levels of extracellular dopamine in dopaminergic terminal fields, suggesting a more complex basis for the psychostimulant reinforcement (Rocha et al., 1998). Besides inhibiting the dopamine transporter, psychostimulants also inhibit the reuptake of serotonin and noradrenaline, which may contribute to their reinforcing actions possibly in part by modulating dopamine neurotransmission (Parsons et al., 1995; Tanda et al., 1997b). Much like psychostimulants, opiate drugs are readily self-administered intravenously by animals, and the systemic and central administration of competitive opiate antagonists will decrease opiate reinforcement (reviewed by Koob and Bloom, 1988; Di Chiara and North, 1992). The reinforcing actions of opiates appear to be largely mediated by the p opioid receptor since selective k antagonists decrease opioid reinforcement in a dosedependent manner (Negus et al., 1993). In addition, morphine reinforcement is abolished in mice with a targeted disruption of the p opioid receptor gene (Matthes et al., 1996). The reinforcing properties of opiates utilize the same circuitry implicated in the actions of cocaine and amphetamine stimulants but may involve additional sites of interaction (Koob and Bloom, 1988) (Table 1). Blockade of opioid receptors either in the VTA or the nucleus accumbens will decrease heroin self-administration. Furthermore, rats will lever press to administer opioid peptides in their nucleus accumbens or VTA, and opiate administration into these restricted brain regions will reinforce drug-seeking behavior (reviewed by Di Chiara and North, 1992; Shippenberg et al., 1992). Opiates, like other drugs of abuse, increase dopamine release in the nucleus accumbens (Di Chiara and Imperato, 1988; Pontieri et al., 1995) (see below). However, the reinforcing effect of opiates in the nucleus accumbens persists when all dopamine projections there are destroyed, suggesting that their reinforcing actions may involve both a dopamine-dependent (VTA) and a dopamine-independent (nucleus accumbens) mechanism (Koob and Bloom, 1988). Sedative-hypnotics, including ethanol, are thought to produce their reinforcing actions through multiple neurotransmitter systems (Engel et al., 1992) (Table 1). One of the major sites proposed for ethanol reinforcement is modulation of GABA receptors (Liljequist and Engel, 1982; Samson and Harris, 1992). GABA antagonists reverse many of the behavioral effects of ethanol (Liljequist and Engel, 1982; Samson and Harris, 1992). Furthermore, the benzodiazepine RO 15-4513 (termed an inverse agonist because it produces effects opposite to those of typical benzodiazepines) will reverse some of the behavioral effects of ethanol, and dose-dependently reduces oral ethanol self-administration in rats (Samson and Harris, 1992). When potent GABA antagonists are microinjected into the brain, the most effective site to reduce ethanol consumption is the central nucleus of the amygdala (Hyttia and Koob, 1995). Ethanol reinforcement also appears to involve activation of brain dopamine systems. Acutely, ethanol consumption or systemic injection reduces the firing rate Review 469 of pars reticulata GABA neurons, which are thought to exert an inhibitory control over VTA dopaminergic neurons (Diana et al., 1993; Merue and Gessa, 1985), and increases concentrations of dopamine in the extracellular compartment in the nucleus accumbens. Dopamine receptor antagonists injected into the nucleus accumbens reduce lever pressing for ethanol in nondependent rats, and basal extracellular dopamine levels are increased in rats chronically consuming low doses of ethanol (reviewed by Koob et al., 1994b). However, virtually complete 6-hydroxydopamine denervation of the dopamine inputs to the nucleus accumbens fails to alter voluntary responding for ethanol, suggesting that nucleus accumbens dopamine transmission may not be critical for reinforcing actions of ethanol (Koob et al., 1994b). Moreover, manipulations that change brain serotonin synaptic availability can decrease ethanol intake (Sellers et al., 1992). For example, serotonin reuptake blockers decrease ethanol intake but so do antagonists of the serotonin-3 and serotonin-2c receptors, suggesting a complex interaction of serotonin function and ethanol reinforcement (LeMarquand et al., 1994). Opiate antagonists also reduce ethanol self-administration in several animal models (Tabakoff and Hoffman, 1996). The central nucleus of the amygdala is particularly sensitive to this opioid antagonism of oral ethanol selfadministration (Heyser, Roberts, and G. F. K., unpublished data). Double-blind, placebo-controlled clinical trials showed that naltrexone can significantly reduce human ethanol consumption, frequency of relapse, and “craving” for ethanol in detoxified alcoholics, suggesting that the motivation to resume ethanol use may involve opioid systems (Volpicelli et al., 1992). Finally, low doses of ethanol will sensitively inhibit the NMDA subtype of glutamate receptors; in drug discrimination studies, animals substitute glutamate antagonists for ethanol (Tabakoff and Hoffman, 1996). Intravenous self-administration of nicotine is also blocked by dopamine antagonists and dopamine-selective lesions of the nucleus accumbens (Corrigall et al., 1992; Dani and Heinemann, 1996). Nicotine withdrawal can also be produced by opioid antagonists (Dani and Heinemann, 1996). Nicotine thus may activate both the dopamine system and some opioid peptide neurons in the same neural circuitry associated with other drugs of abuse (Corrigall et al., 1992) (see Table 1). Exactly which type of nicotinic receptor subunit configuration mediates the reinforcing effects of nicotine is not clear, but neurons in the VTA and nucleus accumbens express high levels of the a6, a2, and a3 subunits (Le Novere et al., 1996). Tetrahydrocannabinol (THC) shares effects in animal models of drug reinforcement similar to those of other drugs of abuse (Anthonyet al., 1994). Upon acute administration, THC decreases reward thresholds in rats (Gardner et al., 1988; Lepore et al., 1996), produces a place preference in rats (Lepore et al., 1995), and, as a synthetic THC analog, is intravenously self-administered in mice (Fratta et al., 1997, Sot. Neurosci., abstract). THC binds to the cannabinoid-1 receptor, which is widely distributed throughout the brain but particularly in the extrapyramidal motor system of the rat (Herkenham et al., 1990). THC activates the mesocorticolimbic Table 2. Drug Effects on Thresholds for Rewarding Brain Stimulation Drug Class Psychostimulants (cocaine, amphetamines) Opiates (morphine, heroin) Nicotine Sedative-Hypnotics (ethanol) THC Acute Administration Withdrawal from Chronic Treatment 1 t 1 t 1 1 T 1 T t dopamine system (Chen et al., 1991), and recent data suggest that THC can selectively increase the release of dopamine in the shell of the nucleus accumbens similar to other drugs of abuse (Tanda et al., 1997a). Negative Reinforcement Associated with Addiction Repeated drug use is thought to arise from the neurochemical actions causing the positive reinforcing effects of a drug. However, the transition from occasional drug use to drug addiction has been thought to require an additional source of reinforcement, the reduction of the aversive (negative) emotional state arising from repeated use. Here, drug taking presumably removes the dysphoria, anxiety, irritability, and other unpleasant feelings produced by drug abstinence. Other somatic physical signs, such as tremor, temperature changes, and sweating, which also reflect the state of dependence, presumably have little if any motivating properties on drug use (Koob, 1996; Koob and Le Moal, 1997). Indeed, one of the defining features of drug addiction is thought to be the establishment of such a negative emotional state (Russell, 1976). Consistent with this hypothesis, all major drugs of abuse have been found to produce a negative emotional state in dependent humans during acute abstinence. The combination of the positive reinforcing effects of the drugs with reduction of the negative emotional states of drug abstinence provides a powerful motivational force for the compulsive drug taking that characterizes addiction. One likely mechanism for this negative emotional state may be a reduction in brain reward function. In studies employing intracranial self-stimulation behavior, to directly study brain reward circuits, animals that have been made chronically dependent show increased reward thresholds (i.e., decreased reward) during withdrawal (reviewed by Koob et al., 1993; Koob, 1996; Koob and Le Moal, 1997). These decreases in reward have been observed following the withdrawal from psychomotor stimulants, opiates, ethanol, THC, and nicotine and are dose related to the amount of drug that had been administered before withdrawal (Epping-Jordan et al., 1998b; Gardner and Vorel, 1998) (Table 2). Common Neuropharmacological Elements in Addiction The molecular and cellular basis for these changes in motivation to take drugs may reside in the neuroadaptations arising in the same neural elements that mediate Neuron 470 Table 3. Neurotransmitters Implicated in the Motivational Effects of Withdrawal from Drugs of Abuse 1 1 1 1 t Dopamine Opioid peptides Serotonin GABA Corticotropin-releasing factor the acute reinforcing actions of these drugs. Earlier work suggested that these adaptations occur both within the drug-sensitive reinforcement system and in additional systems outside the drug-sensitive reward system (Koob and Bloom, 1988). Several common elements have been identified that appear to change with chronic drug administration and could underlie (or mediate) the compulsive self-administration of all drugs (Koob, 1992; Koob, 1996; Koob and Le Moal, 1997) (Table 3). Functional changes in the mesolimbic dopamine systern appear to be common to the chronic actions of all drugs of abuse (Nestler, 1996). Drug discontinuation is accompanied by biochemical and electrophysiological evidence of decreases in dopamine function (Diana et al., 1993; Nestler, 1996; Weiss et al., 1996). During withdrawal from chronic ethanol administration, there is a decrease in extracellular dopamine in the nucleus accumbens, and, concomitantly, neurons of the mesolimbit dopamine system show dramatic reductions in spontaneous firing rates and patterns but not in the number of spontaneously active neurons (Diana et al., 1993; Weiss et al., 1996). These biochemical and electrophysiological changes can be reversed by ethanol administration. Another neurotransmitter system implicated in the acute reinforcing actions of drugs of abuse whose function is altered during the development of dependence is the opioid peptide system, as seen with either chronic opiate usage (Self and Nestler, 1 995; Nestler, 1996) or with other drugs of abuse (Di Chiara and North, 1992). Here, the functional changes are manifest by a dramatic increased sensitivity to opioid receptor antagonists, probably mediated by alterations in opioid receptor signal transduction (see below) (Nestler, 1996; Widnell et al., 1996). In addition, several neurotransmitter systems that are not involved in the acute reinforcing effects of drugs of abuse do appear to become involved following chronic drug administration. These include several neuropeptides, notably dynorphin, neuropeptide FF (NPFF), and CRF. Neurons containing the opioid peptide dynorphin in the nucleus accumbens appear to be functionally activated following chronic administration of cocaine (Hyman, 1996), while anti-opioid neuropeptides such as NPFF may be activated following chronic opiates (Malin et al., 1990; Lake et al., 1992). CRF, with its many actions on hormonal and behavioral responses to stressors, may be a brain system engaged during drug dependence by all drugs of abuse. Activation of the pituitary adrenal axis long has been recognized as a characteristic of drug dependence and withdrawal in humans (Kreek, 1987). Rats treated repeatedly with cocaine, nicotine, and ethanol show significant anxiogenic-like responses following cessation of chronic drug administration that are reversed with intracerebroventricular administration of a CRF antagonist. Microinjections into the central nucleus of the amygdala of lower doses of a CRF antagonist also reversed the anxiogenic-like effects of ethanol withdrawal. Similar doses of the CRF antagonist injected into the amygdala reversed the aversive effects of opiate withdrawal (Koob, 1996). In vivo microdialysis studies have shown an increase in extracellular CRF during ethanol, cocaine, and THC withdrawal (Merlo-Pith et al., 1995; Rodriguez de Fonseca et al., 1997; Richter and Weiss, personal communication). Thus, CRF activation may be a common element in the development of drug dependence and may contribute to motivational effects involving such subjective symptoms as increased stress and negative affect (Koob, 1996). The neurochemical changes highlighted above illustrate neuroadaptations common to all drugs of abuse (Koob, 1996). However, even more intriguing is the possibility that a neuroanatomical entity termed the extended amygdala (Heimer and Alheid, 1991) (Figure 1) may represent a common anatomical substrate for acute drug reward and the negative effects of compulsive drug administration on reward function. The extended amygdala is comprised of the medial subregion of the nucleus accumbens (termed the shell of the nucleus accumbens), the bed nucleus of the stria terminalis, and the central nucleus of the amygdala (Heimer and Alheid, 1991). Heimer has noted that all of these regions share certain cytoarchitectural and circuitry similarities (Heimer and Alheid, 1991). The extended amygdala receives numerous afferents from limbic structures, such as the basolateral amygdala and hippocampus, and sends not only efferents to the medial part of the ventral pallidum but also a large projection to the lateral hypothalamus, thus further defining the specific brain areas that interface classical limbic (emotional) structures with the extrapyramidal motor system. The structures comprising the extended amygdala may further define the neural substrates for the acute reinforcing actions of drugs of abuse (Koob, 1992; Koob, 1996). Acute administration of all the major drugs of abuse produces increases in extracellular levels of dopamine in the shell bf the nucleus accumbens (Pontieri et al., 1995). The ventromedial shell of the nucleus also expresses high levels of the dopamine D3 receptor mRNA (Diaz et al., 1995), and the shell of the nucleus accumbens is particularly sensitive to the cocaine antagonist activity of a dopamine Dl antagonist (Caine et al., 1995). The central nucleus of the amygdala also has a role in ethanol reinforcement. Microinjection of GABA antagonists or opioid peptide antagonists into the central nucleus can attenuate lever pressing for oral ethanol (Hyttia and Koob, 1995; Heyser, Roberts, and G. F. K., unpublished data). Perhaps more intriguing is recent evidence that the extended amygdala may be an important substrate for the changes in the reward system associated with dependence. In animals dependent on ethanol, microinjections of previously ineffective doses of a GABA agonist into the central nucleus of the amygdala decreased ethanol self-administration (Roberts et al., 1996), suggesting that the GABAergic system has been altered to become more responsive to agonists during the course of dependence. Thus, the same neurochemical components in the extended amygdala involved in acute drug actions may become compromised Review 471 Effects of Drugs of Abuse on Sub regions of the Extended Amygdala Nucleus Accumbens Core Nucleus Accumbens Shell (2ocaine Amphetamines wEttland NiCOth? 7HC An&ior Commissure Ethanol wf= NiiWtilW Figure 1. Horizontal Section of a Rat Brain Depicting the Principal Structures of the Extended Amygdala These structures include the central nucleus of the amygdala, the shell part of the nucleus accumbens, and the bed nucleus of the stria terminalis. The drugs listed below each structure refer to potential sites of action of drug reinforcement during the addiction cycle, either positive or negative. Redrawn with permission from Heimer and Alheid (1991). during the development of dependence. One may speculate that additional neurochemical systems also may be engaged within the neurocircuitry of the extended amygdala (Koob and Bloom, 1988), in an attempt to overcome the chronic presence of the perturbing drug and to restore normal function despite the presence of drug. The changes in CRF on the central nucleus of the amygdala observed in dependent animals during withdrawal support this hypothesis (Koob et al., 1994a, 1994b) (see Table 1, Table 2, and Figure 1). Molecular, Cellular, and System Adaptations Associated with Brain Motivational Systems In addition to the changes in neurochemical systems that are common to the chronic administration of drugs of abuse, there are molecular changes that may provide not only the mechanism for the above neurochemical changes but also the substrate for prolonged adaptation to chronic drug administration. Differences in adaptive responses at the molecular level may account for individual differences in vulnerability to dependence to different drugs. The challenge for future research is to link these changes in plasticity to motivational actions of dependence (Figure 2). Several molecular consequences of motivationally important adaptations to chronic cocaine or amphetamine administration have been identified. For example, activation of Dl-like receptors stimulates a cascade of events, including activation of G, proteins and increased intracellular cyclic AMP formation that ultimately may lead to phosphorylation of transcription factors such as cyclic AMP response element-binding protein (CREB) and to the induction of immediate-early genes (see Self and Nestler, 1995; Hyman, 1996) (see Figure 3). An important role for the Dl receptor-CAMP-CREB pathway in the neuroadaptation to chronic drug dependencies is supported also by the recent evidence of effective anti-cocaine actions from dopamine Dl antagonists and effective anti-cocaine priming effects from Dl agonists (i.e., ability of cocaine to reinstate responding in rats that have stopped responding after cocaine has been replaced by saline) (Self et al., 1996). Activation of Gi proteins, linked to D2 receptors, also appears to be involved in the acute effects of cocaine since pertussis toxin, which inactivates Gi proteins, produces a dopamine receptor antagonist-like effect on cocaine selfadministration (Self et al., 1994). In the VTA, repeated administration of cocaine produces transient decreases in Gi proteins that may lead to 02 receptor subsensitivity (Nestler, 1994; Self and Nestler, 1995). More prolonged effects that persist up to one month in the nucleus accumbens include a supersensitivity to Dl -mediated responses (Henry and White, 1991), increased levels of adenylyl cyclase and protein kinase A (PKA), decreased levels of G proteins (Self and Nestler, 1995; Nestler, 1996), and a decrease in the ability of cocaine to induce the immediate-early gene c-f&, followed by the sustained expression of AP-1 transcription factor complexes with altered composition (see below). Chronic administration of cocaine also decreases the levels of neurofilament proteins in the VTA (Self and Nestler, 1995). Lower levels of neurofilament proteins are associated with decreased axonal transport, and this could decrease the amount of tyrosine hydroxylase transported from the VTA to the dopamine nerve terminals in the nucleus accumbens (Self and Nestler, 1995). . ” Neuron 472 Figure 2. Schematic Diagram Showing the Relationship between Different Levels of Analysis in the Study of Addiction and the Role of Neuroadaptive Processes Neuroadaptive mechanisms have been hypothesized to contribute to compulsive behavior and addiction by acting at different levels of the spiraling cycle of the development of dependence (Koob and Le Moal, 1997). Both sensitization and counteradaptation may contribute to changes in hedonic responsiveness and set-points. Such changes could be responsible for short-term reductions in extracellular levels of dopamine release during drug withdrawal (Weiss et al., 1992), which then could trigger upregulation of the cyclic AMP system (Self and Nestler, 1995) (see above). These adaptations within the mesolimbic dopamine system, and its receptor systems, not only could change the function of the dopamine system itself but also may trigger a second major action, namely increased expression of protachykinin and prodynorphin mRNAs. Dynorphin peptides in the nucleus accumbens, in turn, may decrease dopamine release via a presynaptic action on K opioid reCeptOrS; K XJOniStS are known t0 produce aversive effects in rodents and humans (Hyman, 1996). Thus, chronic administration of psychomotor stimulants would induce prodynorphin gene expression in the nucleus accumbens that opposes the effect of cocaine on reward. Chronic opiate administration is associated with little evidence of changes in opioid peptide activity or changes in the number of any of the known opioid receptors (but see Mansour et al., 1995; Zadina et al., 1997). However, there is strong evidence that a dramatic enhancement of sensitivity to the aversive effects of opioid antagonists can occur in brain areas implicated in the acute reinforcing effects of opiates (Stinus et al., 1990; Self and Nestler, 1995; Nestler, 1996). The molecular basis for this effect may be at the level of signal transduction (see Figures 2 and 3). Acute administration of morphine decreases adenylate cyclase activity in the nucleus accumbens, whereas chronic morphine treatment is associated with increases in second messenger systems, including adenylate cyclase activity and PKA (Nestler, 1996). The hypothesis that these effects contribute to the motivational effects of opiates (e.g., tolerance and dependence) is supported by studies showing that direct administration into the nucleus accumbens of agents that inhibit Gai or activate protein kinase decreases the reinforcing effects of opiates (Self et al., 1994). Chronic morphine administration also decreases the level of the transcription factor CREB in the nucleus accumbens. This provides a possible mechanism for morphine to mediate the alterations in gene expression that may underlie the long-term changes in motivational systems associated with opioid dependence (Widnell et al., 1996) (Figure 3). Genetic disruption of two of the three types of CREB in mice dramatically reduced symptoms of morphine withdrawal and produced some reduction in tolerance to the analgesic effects of morphine (Maldonado et al., 1996). Chronic ethanol exposure has been shown to compromise dopamine and GABAergic systems that are linked to the continued desire to consume ethanol (see above). At the molecular level, chronic ethanol is associated with decreases in the ability of ethanol to potentiate GABA-stimulated Cl- flux, decreased expression of the CXI subunitof the GABA complex (Tabakoff and Hoffman, 1996) and other subunits, and increased expression of the p subunit (Tabakoff and Hoffman, 1996). Chronic ethanol also is associated with increases in specific subunits (NRI and NR2A) of NMDA receptors (Tabakoff and Hoffman, 1996). The relationship of these molecular changes in ethanol-receptive elements to the motivational substrates outlined above may ultimately involve changes in the same transduction systems observed for other drugs of abuse (Figures 2 and 3). A possible anatomical substrate for the molecular action of GABA on motivational aspects of ethanol dependence is the amygdala, a brain region considered important in mediating emotional behavior in general. GABAergic neurons in the central amygdala also have been proposed to mediate rapid changes in autonomic activity (Sun and Cassell, 1993), and acute ethanol induces expression of the immediate-early gene c-fos, a marker of neuronal activation, in GABAergic neurons in the central amygdala (MoralesCriado, and P. P. S., unpublished data). Adaptation to chronic nicotine administration also may occur in the same neuronal circuits associated with its initial molecular actions, namely nicotine on nicotinic acetylcholine receptors located in the brain mesolimbic dopamine system. One recent hypothesis seeks to explain nicotine tolerance and dependence on the basis of a prolonged stability in the desensitized state of the nicotinic acetylcholine receptor following the initial stimulation (or “activation”) (Dani and Heinemann, 1996). Consistent with this desensitization hypothesis, longterm nicotine exposure causes an increase in the actual number of nicotinic acetylcholine receptors (Collins et al., 1990). Dani and Heinemann (Dani and Heinemann, 1996) have proposed that nicotine stimulates the mesolimbic dopamine system via activation of nicotinic acetylcholine receptors to produce the acute reinforcing effects of nicotine; with continued use, the inactivation of these receptors by desensitization would then lead to adaptive tolerance. During abstinence, nicotine levels fall, and the increased nicotinic acetylcholine receptors, throughout the brain, begin to recover to a responsive state that may be dependent on the receptor subtype. Engaging nicotinic receptors in non-reward-related pathways . . Review 473 Llgand-gated ion channels 0 Gprotein-coupled receptors Opioids,DA Anandamide, 0 Ion channels Figure 3. Molecular Mechanisms of Neuroadaptation K+ Drugs of abuse, by acting on neurotransmitter systems, affect the phenotypic and functional properties of neurons through the genplasma eral mechanisms outlined in the diagram. membrane Shown are examples of ligand-gated ion channels (1) such as the GABAn and the glutamate NMDA receptor (NMR) and G proteincoupled receptors (R) such as opioid, dopamine (DA), or the cannabinoid CBl receptors, among others (2). The latter is activated by endogenous cannabinoids such as anandamide. These receptors modulate the levels of second messengers like CAMP and Caz+ Changes in (3), which in turn regulate the activity of procetlular tein kinase transducers (4). Such protein kiexcitability Changes ih nases affect the functions of proteins located neurotransmitter in the cytoplasm, plasma membrane, and nuresponses cleus (5-8). Among membrane proteins afpathweyr (PKA,PKC,CaMK,MAPK,etc.) fected are ligand-gated and voltage-gated I I I i.... ion channels (6 and 7). Ethanol, for instance, has been proposed to affect the GABAA response via PKC phosphorylation and, at least in Purkinje cells, via PKA phosphorylation (Tabakoff and Hoffman, 1996). G, and G, proteins also can regulate potassium and calcium channels directly through their 87 subunits (9). Protein kinase transduction pathways also affect the activities of transcription factors (8). Some of these factors, like CREB, are regulated posttranslationally by phosphorylation; others, like Fos, are regulated long-term transcriptionally; still others, like Jun, are set point changes regulated both posttranslationally and/or Itranscriptionally. While membrane and cytoplasmic changes may be only local (e.g., dendritic domains or synaptic boutons), changes in the activity of transcription factors may result in long-term functional changes. These may include changes in gene expression of proteins involved in signal transduction (10) and/or neurotransmission (1 l-l 3), resulting in altered neuronal responses. For example, chronic exposure to psychostimulants or opiates has been reported to increase levels of PKA (10) and adenylyl cyclase (11) in the nucleus accumbens and to decrease levels of Gai (11) (Self and Nestler, 1995; Nestler, 1996). Moreover, chronic ethanol induces differential changes in subunit composition in the GABA, and in the glutamate inotropic receptors (12) and increases expression of voltage-gated calcium channels (VGCC) (13) (Tabakoff and Hoffman, 1996). Chronic exposure to drugs also alters the expression of transcription factors themselves (14). CREB expression, for instance, is depressed in the nucleus accumbens and increased in the locus coeruleus by chronic morphine treatment (Nestler, 1996; Widneil et al., 1996), while chronic cocaine and other chronic treatments induce a transition from Fos induction to the induction of the longer-lasting Fos-related antigens (FRAs) (reviewed by Hyman, 1996). The receptor systems depicted in the figure may not coexist in the same cells. The systematic elucidation of their fine distribution and colocalization as well as the definition of the role of pre- and postsynaptic mechanisms are current and future challenges in the neuroscience of addiction. GABA Glutamate GABA i sfgrrrl trsretiuctivrc i ! --....“....W ..........1_..” .......I .........1...1”..“..._“” could contribute to the aversive emotional states associated with nicotine withdrawal. According to this hypothesis, smokers would be, in effect, ultimately medicating themselves with nicotine to regulate the number of functional nicotinic acetylcholine receptors (Dani and Heinemann, 1996). Recent work shows that such changes may be evident in the OL subunit family, with the (r4, (~2, and a7 subunits showing inactivation with chronic nicotine but the a3 subunit (which is similar in structure to the a6 subunit) showing resistance to desensitization (Olale et al., 1997). Thus, one could speculate that there are two powerful forces for development of nicotine addiction: desensitization of nicotinic receptors in non-reward pathways (self-medication) and resistance to desensitization in reward pathways (positive reinforcement). Marijuana is a drug of abuse and addiction in humans, and chronic high dose administration of THC or THC analogs produces a dependence syndrome as measured by behavioral and subjective signs of withdrawal (Aceto et al., 1995). In animals, a withdrawal syndrome is precipitated by administration of competitive THC antagonists to animals treated chronically with cannabinoids (Aceto et al., 1995), and this precipitated cannabinoid withdrawal produced activation of limbic structures as measured by c-fos activation and an increase in extracellular levels of CRF in the central nucleus of the amygdala as seen by other major drugs of abuse (Rodriguez de Fonseca et al., 1997). Whether chronic THC produces cellular and molecular changes in the extended amygdala similar to other drugs of abuse will be a challenge for future research. Craving and Relapse Because drug addiction is a chronic relapsing disorder, the study of vulnerability to relapse is a particularly compelling challenge. However, the biological basis for the states of craving and protracted abstinence are difficult to define. Vulnerability to reinstatement of drug-taking Neuron 474 behavior and ultimately a continuation of compulsive drug use presumably reflects some underlying prolonged perturbation that emerges long after the withdrawal reactions have subsided. A residual deficit state in the reward system, or sensitization of the reward system to stimuli that predict drug effects, or both, could be responsible for this vulnerability (Koob and Le Moal, 1997) (Figure 2). Animal models of drug craving and relapse continue to be developed and refined and are based largely on conditioned reinforcement where previously neutral stimuli, such as drug injection paraphernalia or specific locations where ’ drugs are obtained, have become paired with the drug state or the drug withdrawal state (Koob, 1995). The neural substrates for such conditioned positive reinforcement may involve elements of the extended amygdala and its afferent circuits from the basolateral amygdala (Everitt et al., 1991) and the mesolimbic dopamine system. The neural substrates for any hypothetical conditioned negative reinforcement are largely unknown. However, by definition, any conditioning that occurs during chronic drug use also involves the formation of subtle associations, but it is unknown whether the associations formed by the pairing of drugs with previously neutral stimuli utilize the same circuits as those for memories not related to drugs. A possible molecular mechanism for such learned, long-term neuroadaptation may involve alterations to gene expression (Figure 3). For example, Nestler and coworkers have observed that acute cocaine induces the transcription factor complex AP-1 in the striatum and nucleus accumbens. With continued exposure to drugs, the composition of AP-1 gradually changes from one in which the immediate early-gene product, c&s, is present, to one in which a longer-lived, Fos-related antigen (termed “chronic FRA”) substitutes for c-fos (Hope et al., 1994). Because of the longer half-life of chronic FRA, repeated exposure to cocaine leads to its progressive accumulation (Hope et al., 1994). Shifts of AP-1 composition in relevant CNS regions may represent a general neuroadaptive process that contributes to long-term functional changes (Rossetti and Carboni, 1995). Nicotine or cocaine self-administration also has been shown to elicit overlapping patterns of c-fos induction followed by induction of FRA immunoreactivity in the nucleus accumbens and other regions, but not in the amygdala (Merlo-Pith et al., 1997). A recent study showed c-fos induction in the nucleus accumbens, central amygdala, and bed nucleus of the stria terminalis following acute exposure to a synthetic cannabinoid agonist (Rodriguez de Fonseca et al., 1997). The differences in activation patterns of immediate-early genes and related proteins, such as the FRAs, by different classes of drugs within neurons of the extended amygdala may underlie their specific mechanisms of action. Taken together, these observations support the potential contribution of alteration in gene expression to the long-term neuroadaptive changes associated with the motivational aspects of drug dependence. However, of critical importance will be the correlation over time of the specific biochemical changes that account for increased vulnerability for drug seeking in defined components of all aspects of the addiction cycle: acquisition, maintenance, withdrawal, and relapse. A particular challenge for future studies in the neuroscience of addiction will be to elucidate the neuroadaptive changes produced by chronic drug use in animal models of protracted abstinence and relapse. Presumably, the answers will be found not only in the same molecular and cellular elements of the neurochemical systems and neurocircuitry responsible for the positive and negative reinforcement associated with chronic drug use, but also in the multiple neuroadaptive mechanisms that establish long-term memories of the drug rewards (Bailey et al., 1996). Acknowledgments This is publication number 11130-NP from The Scripps Research Institute. Research was supported by National Institutes of Health grants DA04043, DA04398, and DA08467 from the National Institute on Drug Abuse and AA06420 and AA08459 from the National Institute on Alcohol Abuse and Alcoholism. The authors would like to thank Mike Arends for his valuable assistance with manuscript preparation. 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