Over the past 20 years, drug abuse research has contributed to impressive gains in the neurosciences and in our understanding of brain function. Neuroscience research as it relates to drug abuse has advanced knowledge about neurotransmitters and neural pathways, and has yielded information about brain mechanisms both under normal conditions and when affected by drugs of abuse. That knowledge has already been translated into improved clinical care and has had significant impacts on other scientific disciplines.
The goal of neuroscience research in the area of drug dependence is to determine the actions of abusable drugs on the brain that result in dependence and to determine the neural substrates that make one individual inherently vulnerable to such actions and others relatively resistant. That knowledge can have an impact on the ways in which drug abuse and dependence are managed clinically and on the way they are viewed by our society. Neuroscience research can add to the knowledge base in the science of addiction and provide information for the development of more effective medications to treat drug dependence. New pharmacotherapies will significantly improve the effectiveness of psychosocial interventions. It must be emphasized that it is impossible to predict all of the benefits of ongoing fundamental neuroscience research in the drug abuse field. Many of the advances that will be discussed throughout this chapter were unanticipated, yet clearly improved public health in many ways.
The interface between basic neurobiology and the applied neuroscience of drug abuse research has been a rich and fruitful part of the approach termed integrative neuroscience. Drug abuse research has con-
tributed to many discoveries in neuroendocrinology and the neurobiology of stress including the discovery of opioid peptides and stress neurotransmitters, the neurochemical control of stress hormone, and reproductive hormone release. In addition, drug abuse research impacts on disciplines as diverse as molecular biology, the neurobiology of emotional behavior, and the neurobiology of cognitive function in the effort to understand the complex phenomena associated with a course of drug dependence.
The following chapter contains a technical overview illustrating the complexity of the neurotransmission processes involved in the neurobiology of drug dependence, a description of the many advances in understanding the neurobiological basis for drug dependence, a summary of gaps and needs, and finally recommendations for future research. The technical overview provides the vocabulary and basic concepts necessary to understand how drugs can interact at many different functional levels including the molecular, cellular, and systems levels. The section on accomplishments details the significant advances in understanding the neurobiology of drug reinforcement and the beginnings of our understanding of the processes of neuroadaptation to these systems associated with dependence. In addition, the chapter describes progress in human imaging research and the recent developments in understanding brain mechanisms of pain and analgesia. Gaps and needs are identified that focus on the chronic consequences of drug exposure in brain systems implicated in the motivational effects of drug dependence at the molecular, cellular, and system levels of analysis. Finally, the chapter identifies numerous areas for research opportunities that will aid in our understanding of the neurobiology of drug dependence and help integrate this basic research with the applied problems of vulnerability, treatment, and prevention of drug abuse. These areas include molecular neurobiology, genetics research, animal models of dependence, brain imaging, co-occurring psychiatric disorders, HIV models, neurotoxicity of drug dependence, immunology, analgesia and pain, and relapse and prolonged abstinence.
NEUROTRANSMISSION AND ITS EFFECTS
The human brain is composed of an enormous number of neurons, with estimates ranging from 10 billion to 10 trillion (reviewed by Kandel et al., 1991; Hyman and Nestler, 1993). These neurons are organized in such a way that they communicate with one another in a highly intricate and specific manner. This process of communication is referred to as synaptic transmission.
In a simplified scheme, neurons consist of a cell body or soma; mul-
tiple dendrites that arise from the cell body to receive incoming signals; and usually a single axon that also arises from the cell body. Axons can be very long and give rise to outgoing signals through their branched ends (terminals). A single neuron can possess thousands of axon terminals and thereby form connections (called synapses) with up to thousands of other neurons. The brain utilizes a chemical process of neurotransmission to transfer information across synapses. Briefly, an electrochemical impulse produced by changes in concentrations of ions across the axon membrane travels down the axon of one neuron, invades the axon's nerve terminals, and triggers the release of a chemical substance, called a neurotransmitter, from the terminals. The neurotransmitter diffuses across the synaptic cleft (the space between the two neurons) and binds to specific receptor proteins located on the surface of the cell, or plasma membrane, of the next neuron. The binding of a neurotransmitter to its receptor activates the receptor and causes a change in the flow of ions across the cell membrane, which can either lead to or inhibit the generation of electrical impulses in that next neuron. The neurotransmitter stimulus is then "turned off" either by enzymatic degradation in the synaptic cleft or by proteinmediated reuptake of neurotransmitter into the nerve terminal. Neurons receive incoming signals from hundreds or thousands of nerve terminals. Whether a neuron fires an impulse is determined by the summation of those numerous inputs.
Neuronal membranes contain classes of proteins, termed ion pumps, that maintain unequal concentrations of ions (e.g., Na+, K+, Ca2+, C1-) between the outside and inside of the cell. The most important pump is termed the Na+-K+ ATPase (adenosine triphosphatase). Neurons are polarized, meaning that the inside of the cell is negatively charged with respect to the outside. Neurons also possess other proteins in their plasma membrane, termed ion channels, that allow passage of specific ions across the cell membrane. Neurotransmitters regulate the electrical properties of neurons by activating or inhibiting the activity of specific types of ion channels.
Neurotransmitters and Their Receptors
The majority of neurotransmission in the brain is performed by amino acid neurotransmitters, which are contained in two-thirds of all synapses in the brain. Glutamate is the major excitatory neurotransmitter in the brain because its receptor channel permits Na+ (and in some cases Ca2+) to flow into the cell; the major inhibitory neurotransmitter in the brain is gamma-aminobutyric acid (GABA) (GABA's receptor channel carries C1 into the cell).
Most other neurotransmitters in the brain bind to receptor proteins
that do not contain ion channels within their structures. Rather, these receptors produce their physiological effects by interacting with a special class of proteins, called G proteins, which are composed of three variable molecules, called alpha, beta, and gamma subunits. When a neurotransmitter binds to a G protein-coupled receptor, the G protein dissociates into a free alpha and free beta-gamma subunit, which then interacts with many other cellular proteins to produce a variety of physiological effects. For example, specific types of ion channels can be induced to increase or decrease their activity by the action of G protein subunits.
Second Messengers and Protein Phosphorylation
The G protein-coupled receptors also influence many other neural processes through complex pathways of intracellular messengers. The first steps in these pathways are "second messengers" (the neurotransmitter is considered the first messenger, and the G protein a coupling factor). Prominent second messengers in the brain are cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanosine monophosphate), Ca2+, nitric oxide, and metabolites of arachidonic acid (e.g., prostaglandins) and phosphatidylinositol. The G protein-coupled receptors control the levels of these second messengers by regulating the activity of enzymes that catalyze the synthesis and degradation of second messengers, with different effects produced depending on the G protein involved.1 For example, neurotransmitters that increase cAMP levels act through Gs, which binds to and stimulates adenylyl cyclase, the enzyme that catalyzes the synthesis of cAMP. Other neurotransmitters decrease cAMP levels by acting through Gi, which binds to and inhibits adenylyl cyclase. Still other neurotransmitters do not affect cAMP, but instead increase the generation of phosphatidylinositol-derived second messengers.
The next step in these intracellular pathways is the regulation, by second messengers, of protein phosphorylation, the process by which phosphate groups are added to or removed from specific amino acid residues by protein kinases and protein phosphatases, respectively. Phosphate groups, because of their large size and negative charge, affect the conformation and charge of proteins, which in turn affect their physiological function. For example, phosphorylation of ion channels and pumps affects their ability to open or close or to allow ions to pass through them. Phosphorylation of receptors affects their ability to bind to their
neurotransmitters or interact with their G proteins. Phosphorylation of enzymes affects their catalytic activity (e.g., phosphorylation of adenylyl cyclase can increase its capacity to synthesize cAMP).
The brain contains many types of protein kinases and protein phosphatases that exhibit differential regulation. For example, cAMP activates cAMP-dependent protein kinases, Ca2+ activates Ca2+-dependent protein kinases, etc. Each type of protein kinase then phosphorylates a specific array of target proteins and thereby produces many additional effects of the original neurotransmitter-G protein-second messenger stimulus.
Due to the multiple effects of phosphorylation on a number of important intracellular processes, a neurotransmitter stimulus can influence virtually every chemical process that occurs within its target neurons. Some effects, such as alterations in electrical activity, are very rapid (within seconds) and short-lived. Other effects, such as alterations in gene expression, can develop more slowly (over minutes or hours) and last for a long time. These more long-lasting effects of a neurotransmitter stimulus alter the manner in which the target neuron responds to subsequent stimuli—both the original neurotransmitter and others—and presumably represent the basis of neural adaptation and change, called plasticity. Together, these types of responses of widely differing time courses allow neurons to exert very complex control over other neurons operating within neural circuits.
Neurotrophic Factor Signaling Pathways
Second-messenger–regulated protein phosphorylation is just one component of a neuron's complex intracellular regulatory mechanisms. Neurons contain many protein kinases and protein phosphatases in addition to those regulated by second messengers, and these enzymes also contribute to the diverse effects that a neurotransmitter stimulus exerts on its target neurons. For example, neurotrophic factors were first studied for their important role in neural development and differentiation. However, it is now known that neurotrophic factors also play an important role in the regulation of the fully differentiated adult brain. One important family of neurotrophic factors, called neurotrophins, binds to a class of receptor that contains a special type of protein kinase within its structure, a protein tyrosine kinase, which phosphorylates proteins specifically on tyrosine residues. Binding of neurotrophin to its protein tyrosine kinase receptor activates the kinase activity and leads to the phosphorylation of specific cellular proteins and, eventually, to a cascade of protein kinase activity. Thus, neurotrophic factor-related signaling pathways are another example of the complexity of a neuron's intracellular regulatory machinery, and serve to highlight the complex types of effects that a
neurotransmitter stimulus produces in its target neurons which ultimately contributes to the short- and long-term effects of neurotransmitters on the brain.
Understanding Drug Dependence in the Context of Neurotransmission
All drugs of abuse interact initially with receptor or reuptake proteins, summarized in Table 3.1 (Nestler et al., 1995). For example, opiates activate opioid receptors, and cocaine inhibits reuptake proteins for the monoamine neurotransmitters (which include dopamine, norepinephrine, and serotonin). These initial effects lead to alterations in the levels of specific neurotransmitters, or to different activation states of specific neurotransmitter receptors, in the brain. Opiate activation of opioid receptors, for example, leads to recruitment of inhibitory and related G proteins. This, in turn, leads to activation of K+ channels and inhibition of Ca2+ channels. Both are inhibitory actions, because more K+ flows out of the cell and less Ca2+ flows into the cell. Thus, the electrical properties of the target neurons are affected relatively rapidly by opiates. Recruitment of the inhibitory G protein also inhibits adenylyl cyclase, and reductions in cellular Ca2+ levels decrease Ca2+-dependent protein phosphorylation cascades, altering the activity of still additional ion channels. These effects, along with changes in many other neural processes within target neurons, contribute further to the acute effects of opiates. The sum of such
TABLE 3.1 Acute Effects of Abused Drugs on Neurotransmitters
Agonist at opioid receptors
Inhibits monoamine reuptake transporters
Stimulates monoamine release
Facilitates GABAA receptor function and inhibits
N-methyl-D-aspartate (NMDA) glutamate receptor functiona
Agonist at nicotinic acetylcholine receptors
Agonist at cannabinoid receptorsb
Partial agonist at 5-HT2c serotonin receptors
Antagonist at NMDA glutamate receptors
a The mechanism by which alcohol produces these effects has not been established but would not appear to involve direct alcohol binding to the receptors as is the case for the other drugs listed in this table.
b Although a specific receptor for cannabinoids has been identified in the brain, the endogenous ligand for this receptor has not yet been identified with certainty.
changes presumably triggers the longer-term effects of the drugs that eventually lead to abuse, dependence, tolerance, and withdrawal.
Significant advances in understanding the neurobiological basis of drug dependence in the past 25 years are now beginning to provide a strong scientific basis for drug abuse treatment, prevention, and etiology. Drug dependence has long been associated with some perturbation of the brain reward systems. At the systems level, specific neural circuits within the midbrain-forebrain connection of the medial forebrain bundle have been identified that mediate the acute reinforcing effects of drugs (Figure 3.1) (Koob, 1992a). These neural circuits are composed of specific chemical neurotransmitters and include the midbrain dopamine systems, the endogenous opioid peptide systems, and other neurotransmitters such as serotonin, GABA, and glutamate. These systems appear to be modified during the development of dependence and appear to remain sensitive to future perturbations. Cellular studies have identified specific changes in the function of different components of that midbrain-forebrain system and are beginning to provide a framework for the adaptive changes within neurons that are associated with withdrawal and sensitization (Nestler, 1992). Molecular studies not only have identified the specific neurotransmitter receptors and receptor subtypes important for mediating those reinforcement actions, but also have begun to provide a molecular basis for the long-term plasticity associated with relapse and vulnerability (Nestler, 1994). The remainder of this section highlights some of the neurobiological advances resulting from research on individual differences; neural substrates of reinforcement, withdrawal, tolerance, and relapse; pharmacotherapy; and brain imaging.
It is widely presumed that individuals differ in their predilection for drug dependence (see Chapter 5). This has been demonstrated in epidemiological studies of alcoholism, but it remains largely unproven for other addictive disorders. There is, however, growing evidence of individual differences in responsiveness to drugs of abuse in laboratory animals.
Genetically inbred strains of mice and rats exhibit clearly different behavioral responses to one or another drug of abuse (Li and Lumeng, 1984; Pickens and Svikis, 1988; George and Goldberg, 1989; Guitart et al.,
1993; Kosten et al., 1994). Such strain differences have been demonstrated with respect to numerous behavioral measures, including locomotor activity and sensitization, physical dependence, drug self-administration, conditioned place preference, and brain stimulation reward (Li et al., 1986; Crabbe et al., 1994). These observations suggest that there are likely genetic determinants of diverse aspects of drug action, including drug reinforcement. Researchers have also observed that genetically inbred strains of mice and rats differ not only in acute responses to drugs of abuse but also in responses to repeated drug exposure (e.g., George and Goldberg, 1989; Nestler, 1992; Guitart et al., 1993; Kosten et al., 1994), indicating that pharmacodynamic differences may reside in part at the level of gene expression. This research has implications for the treatment of drug abuse discussed later in the chapter.
In animal models, environmental factors also contribute to an individual's responses to drugs of abuse. First, exposure to a drug of abuse itself influences an animal's subsequent responses to the drug, including the reinforcing effects of a drug (Piazza et al., 1989; Horger et al., 1992). Second, other types of environmental factors have been shown to influence an animal's responses to drugs of abuse. One prominent example is stress, which can enhance the reinforcing and locomotor activating effects of several drugs of abuse, including cocaine and other stimulants, opiates, and alcohol (Volpicelli et al., 1986; Piazza et al., 1989; Vezina and Stewart, 1990; Cunningham and Kelley, 1992; Hamamura and Fibiger, 1993; Koob and Cador, 1993; Sorg and Kalivas, 1993; Goeders and Guerin, 1994; Shaham and Stewart, 1994). The effects of stress may be mediated, at least in part, via stress systems such as the hypothalamic-pituitary-adrenal axis, which is known to be activated by stress, and extrahypothalmic stress systems because mediators of those systems, including corticotropin-releasing factor (CRF) and glucocorticoids, alter drug reinforcement and drug-induced locomotor activity (Cole et al., 1990; Piazza et al., 1991). These findings have relevance in the clinical setting for the treatment of drug dependence since continued exposure to environmental factors increases an individual's risk for drug abuse and dependence (see Chapter 2). More work is needed, however, in the area of environmental factors on drug dependence and their neurobiological impact.
One way to understand these observations is that genes determine an individual animal's potential responses to drugs of abuse, whereas envi-
ronmental factors shape that genetic potential. That is, environmental exposures (e.g., a drug or stress) alter the brain in different ways depending on the genetic template of the brain. Particularly powerful environmental exposures (e.g., high levels of a drug of abuse) may lead to the same types of changes in the brain despite genetic differences (Nestler, 1992). Together, genetic and environmental factors combine to set an individual's responses to drugs of abuse. Identification of the specific genetic and environmental factors that influence the actions of drugs of abuse in animal models can provide insight into the types of genetic factors that contribute to an individual vulnerability for drug dependence in humans (Hilbert et al., 1991).
Neural Substrates of Drug Abuse
Neural Substrates of Reinforcement
A multineurotransmitter system called the medial forebrain bundle, which courses from the ventral midbrain to the basal forebrain, has long been associated with reinforcement and reward (Olds and Milner, 1954; Olds, 1962; Stein, 1968; Wise, 1989). Electrical stimulation through electrodes implanted along this bundle is considered to be pleasurable or rewarding because animals will perform certain tasks repeatedly (e.g., pressing a bar) to trigger the stimulation (self-stimulation). Thresholds for that intracranial self-stimulation are lowered by drugs of abuse, suggesting that they ''sensitize" the brain reward system. Recent advances exploring the neurobiological basis for the positive reinforcing effects of drugs of abuse have focused on specific neurochemical systems that make up the medial forebrain bundle reward system.
Psychomotor stimulants, such as cocaine and amphetamine, appear to depend on an increase in the synaptic release of dopamine in the mesolimbic dopamine system (Koob, 1992b). This system has its cell bodies of origin in the ventral tegmental area and projects to the nucleus accumbens, olfactory tubercle, frontal cortex, and amygdala. Cocaine is thought to act mainly to block reuptake of dopamine by binding to a specific protein, the dopamine transporter protein, involved in reuptake; amphetamines both enhance dopamine release and block its reuptake. Three of the five cloned dopamine receptor subtypes have been implicated in the reinforcing actions of cocaine (Woolverton, 1986; Koob, 1992b; Caine and Koob, 1993).
Opiate drugs bind to opioid receptors to produce their reinforcing effects.2 The mu receptor appears to be most important for the reinforc-
ing effects of heroin and morphine, and the most important brain sites for the acute reinforcing actions of those drugs appear to be in the ventral tegmental area and the nucleus accumbens. Opiates stimulate the release of dopamine in the terminal areas of the mesolimbic dopamine system, and there also appears to be a dopamine-independent action in the region of the nucleus accumbens on neuronal systems that receive a dopaminergic input (Koob, 1992a).
Ethanol and other sedative hypnotics clearly have multiple sites of action for their acute reinforcing effects, which depend on facilitation of GABAergic neurotransmission, stimulation of dopamine release at low doses, activation of endogenous opioid peptide systems, and antagonism of serotonergic and glutamatergic neurotransmission. The exact sites for these actions are under study but appear again to involve the mesolimbic dopamine system and its connections in the basal forebrain, particularly in limbic areas such as the amygdala.
Nicotine is a direct agonist at brain nicotinic acetylcholine receptors, which are widely distributed throughout the brain. Nicotine self-administration is blocked by dopamine antagonists and opioid peptide antagonists, and both a nicotinic acetylcholine antagonist and an opiate antagonist have been shown to precipitate nicotine withdrawal in rodents (Malin et al., 1993, 1994). Nicotine is thus thought to activate both the mesolimbic dopamine system and opioid peptide systems in the same neural circuitry associated with other drugs of abuse (Corrigall et al., 1992).
The neurobiological substrates for the acute reinforcing actions of psychedelic drugs are less well understood. Indeed, rodents and nonhuman primates will not self-administer psychedelic drugs. Lysergic acid diethylamide (LSD) clearly involves a serotonergic action, possibly as a postsynaptic agonist. However, the brain sites and specific subtypes involved are still under study. Little is known about the neurobiology of the acute reinforcing actions of marijuana, but the cloning of the tetrahydrocannabinol (THC) receptor and the discovery of endogenous THC compounds in the brain offer exciting new approaches to this question, discussed below (Matsuda et al., 1990; Devane et al., 1992).
Neural Substrates for Drug Tolerance
The neural substrates for drug tolerance overlap significantly with those associated with dependence because tolerance and dependence may be components of the same neuroadaptive process. Tolerance also involves associative processes (processes of learning where previously neutral stimuli come to acquire significance through pairing with biologically significant events), however, and the role of associative processes has been most explored in the context of opiate drugs and sedative-hypnotics
such as alcohol (Young and Goudie, 1995). Both operant (behavioral tolerance) and classical (context-dependent tolerance) conditioning have been shown to play a role in drug tolerance, and mechanisms for these associative processes may involve several neurotransmitters independent of their role in dependence. Norepinephrine and serotonin have long been known to be involved in the development of tolerance to ethanol and barbiturates (Tabakoff and Hoffman, 1992). More recently, administration of glutamate antagonists has been shown to block the development of tolerance, again consistent with an associative component of tolerance (Trujillo and Akil, 1991).
Mechanisms of tolerance at the molecular level often overlap with those of dependence (Nestler et al., 1993).3 For example, up-regulation of the cAMP pathway could be a mechanism of tolerance; the changes would be expected to oppose the acute actions of opiates of inhibiting adenylyl cyclase. In addition, tolerance seems to involve the functional uncoupling of opioid receptors from their G proteins. The mechanisms underlying this uncoupling remain unknown but could involve drug-induced changes in the phosphorylation state of the receptors or G proteins that reduce their affinity for each other. Another possible mechanism of tolerance involves drug-induced changes in the ion channels that mediate the acute effects of drugs. For example, alterations in the phosphorylation state, amount, or even type of channel conceivably could contribute to drug tolerance (Nestler, 1992).
Neural Substrates of Withdrawal
Withdrawal from chronic use of drugs of abuse is characterized by a dependence syndrome that is made up of two elements. The objectively observable physical signs of alcohol withdrawal are tremor and autonomic hyperactivity; abdominal discomfort and pain are associated with opiate withdrawal. The self-reported "psychological" signs of drug withdrawal, which may be considered more motivational, are usually different components of a negative emotional state including dysphoria, depression, anxiety, and malaise (Koob et al., 1993) and are difficult to measure directly in animals. Behavioral history is a primary determinant of whether withdrawal and the negative affective state associated with it produce drug-seeking behavior. For individuals with a history of selfmedication of opiates and alcohol, physical dependence is an important
factor in motivating individuals to seek out and self-administer opiates and alcohol. The phenomenon of physical dependence, however, does not produce drug-seeking behavior in the majority of individuals made physically dependent in the course of treatment with an opiate for a medical condition. The neural substrates for the physical signs of withdrawal are, in fact, not well understood. There is evidence that the changes in body temperature associated with opiate withdrawal may be due to interactions in the hypothalamus. The neural substrates for many of the other physical signs are distributed widely throughout the brain. Much evidence implicates the nucleus locus coeruleus (a nucleus containing exclusively norepinephrine neurons located in the brain stem region called the pons) in the activational properties and stresslike effects of opiate withdrawal (Aghajanian, 1978; Taylor et al., 1988; Maldonado et al., 1992). Little evidence exists for the neural substrates of ethanol withdrawal, but some neuropharmacological mechanisms have been identified including a decrease in GABAergic function, an increase in glutamatergic function (Grant et al., 1990; Tabakoff and Hoffman, 1992; Koob et al., 1994b), and related changes in calcium channel function (Littleton et al., 1992).
Additional research has begun to focus on the neural substrates and neuropharmacological mechanisms of the negative affective states associated with drug withdrawal—effects that probably produce much of the negative reinforcement associated with drug dependence. The same neural systems implicated in the positive reinforcing effects of drugs of abuse have been shown to be involved in those motivational effects. Evidence suggests that reward thresholds are increased (decrease in reward) following chronic administration of all major drugs of abuse, including opiates, psychostimulants, alcohol, nicotine, and THC. These effects reflect changes in the activity of the same mesolimbic system (midbrain-forebrain system) implicated in the positive reinforcing effects of drugs and can last up to 72 hours (Schaefer and Michael, 1986; Markou and Koob, 1991; Koob et al., 1993, 1994b; Schulteis et al., 1994, 1995).
The neurobiology of the change in reward function associated with drug dependence is a very active area of current research. Decreases in the function of neurochemical systems associated with the same neurotransmitters implicated in the acute reinforcing effects of drugs have been observed during withdrawal following chronic administration of cocaine, opiates, and ethanol. One example is where dopamine function in the nucleus accumbens appears to be decreased during cocaine, opiate, and ethanol withdrawal as measured by in vivo microdialysis (Weiss et al., 1992). Also, there is evidence of decreased opioid peptide receptor function in the nucleus accumbens during opiate withdrawal (Nestler, 1992). Serotonin function also appears to be decreased during acute withdrawal from psychostimulants and ethanol in the nucleus accumbens
(Parsons et al., 1995). As noted above, both GABAergic and glutamatergic systems have been implicated in ethanol withdrawal and may be of motivational significance for the changes occurring in the mesolimbic system and its connections. All of those transmitter systems have been implicated in the acute reinforcing effects of those drugs of abuse. However, evidence also exists for the recruitment of other neurotransmitter systems associated with stress-like responses during drug dependence. One example is the increased functional activity of the opioid peptide dynorphin in the nucleus accumbens following chronic cocaine administration, and this may contribute to the negative affective state of withdrawal (Hurd et al., 1992; Spanagel et al., 1992). Also, corticotropin releasing factor function appears to be activated during acute withdrawal to cocaine, alcohol, and opiates, and thus may mediate aspects of stress associated with abstinence (Koob et al., 1994a). More prolonged post-acute withdrawal changes have been observed in the mesolimbic dopamine system that may subserve the phenomenon of sensitization. Animals previously exposed to stress or psychostimulant drugs show enhanced responsiveness to the activating and rewarding effects of psychostimulants after the acute withdrawal period. This behavioral sensitization is paralleled by increased dopamine activity in the mesolimbic dopamine system (Robinson and Berridge, 1993).
Significant insight is now available concerning the molecular and cellular mechanisms of drug dependence. A model of such mechanisms is the locus coeruleus, the major noradrenergic nucleus in the brain, which plays an important role in physical dependence on opiates (Aghajanian, 1978; Taylor et al., 1988; Rasmussen et al., 1990; Koob et al., 1992; Maldonado and Koob, 1993). Activation of the locus coeruleus has been shown to mediate many of the signs and symptoms of physical opiate withdrawal in rodents and nonhuman primates. In fact, it was the identification of opiate action in the locus coeruleus that led to the introduction of clonidine, an alpha-2-adrenergic agonist, as the first nonopiate treatment that decreases the autonomically mediated signs of opiate withdrawal (Aghajanian, 1978; Gold et al., 1978).
We now know that the activation of the locus coeruleus during withdrawal is due to a combination of intrinsic (arising within the specific brain region) and extrinsic (arising from another brain region) factors. The intrinsic mechanisms involve up-regulation of the cAMP pathway (Sharma et al., 1975; Nestler, 1992; Nestler et al., 1993). Acutely, opiates inhibit the cAMP pathway in the locus coeruleus by inhibiting adenylyl cyclase, a molecular site of action for opiate neuroadaptation described above (Collier, 1980). In contrast, chronic exposure to opiates increases the amount of adenylyl cyclase and cAMP-dependent protein kinase expressed in the neurons. This up-regulated cAMP pathway has been shown
to contribute to the increase in the electrical excitability of locus coeruleus neurons associated with withdrawal. A major unanswered question is the precise mechanism (e.g., at the level of transcription, translation, or protein modification) by which chronic opiate exposure leads to up-regulation of the cAMP pathway (Nestler, 1992).
The extrinsic mechanisms of withdrawal activation of the locus coeruleus involve increased activation of the major glutamatergic input to the locus coeruleus, which arises from a brain stem area called the paragigantocellularis (Rasmussen and Aghajanian, 1989; Akaoka and Aston-Jones, 1991). A major unanswered question is what drives this increase in glutamatergic tone. Presumably, chronic opiate exposure leads to changes in the glutamatergic neurons of the paragigantocellularis themselves or in neurons that drive those neurons in some neural circuit (Nestler, 1992).
Much less is known about the molecular and cellular mechanisms of changes in the negative affective state associated with drug dependence, although there is some evidence to suggest that similar mechanisms may be involved. Several drugs of abuse up-regulate the cAMP pathway in the nucleus accumbens after chronic administration (Nestler et al., 1993; Nestler, 1994; Self and Nestler, 1995). This up-regulation could mediate some of the documented electrophysiological changes in the nucleus accumbens associated with chronic drug exposure, such as enhanced responsiveness of D-1 dopamine receptors after chronic cocaine treatment (Henry and White, 1991). Moreover, studies involving direct administration of activators or inhibitors of the cAMP pathway into the nucleus accumbens are consistent with the interpretation that up-regulation of the cAMP pathway in this brain region may contribute to the negative affective state during drug withdrawal (Self and Nestler, 1995). The development of improved animal models will enable further study of negative affective states associated with drug withdrawal.
Neural Substrates of Relapse
Neurobiological mechanisms associated with relapse have been hampered by limited development of animal models. The term relapse is often used to describe a return to drug use despite an individual's attempt to remain abstinent. Thus, incorporation of some motivation to remain abstinent in animal models is necessary. The few studies that exist, using neuropharmacological probes to reinstate self-administration in animals trained and then extinguished on intravenous drug self-administration, have shown that drugs that activate the mesolimbic dopamine system rapidly reinstate intravenous self-administration (de Wit and Stewart, 1981; Stewart and de Wit, 1987). Further progress in understanding relapse will require better animal models.
The hypothesis that antagonizing the positive reinforcing actions of drugs of abuse would prevent relapse and effectively treat drug dependence has received significant preclinical attention. Basic neuropharmacology has shown that all the behavioral effects of opiate drugs, including their positive reinforcing actions, can be reversed by the opiate antagonist naloxone (Di Chiara and North, 1992; Koob, 1992a). The opiate receptor subtype involved in heroin and morphine reinforcement appears to be largely the mu receptor. For cocaine, no specific competitive antagonist has been identified, but antagonism of dopamine receptors in the mesolimbic system appears to block competitively the reinforcing effects of cocaine (Woolverton and Johnson, 1992). Three of the five dopamine receptor subtypes have been implicated in these reinforcing effects, particularly the D-1 and D-3 receptors.
For benzodiazepines, a selective competitive antagonist has been characterized, but not studied in the context of benzodiazepine reinforcement, and it has little effect on ethanol reinforcement (Samson and Harris, 1992). Ethanol reinforcement can be blunted by antagonists and agonists to a number of neurotransmitter systems (Samson and Harris, 1992; Koob et al., 1994b), but none to date has proven wholly specific to or competitive with ethanol. Ethanol reinforcement can be decreased by GABA antagonists, dopamine antagonists, serotonin agonists, and opioid antagonists. Opiate antagonist effects appear to involve both mu and delta receptors, which has led to the introduction of naltrexone, the first new pharmacotherapeutic treatment for alcoholism in 40 years. Recent identification of a competitive THC antagonist will most certainly lead to its testing in reinforcement models. However, the clinical value of such an approach clearly still needs to be established, given the very limited success of opiate antagonists in treating opiate dependence.
Work on the development of antagonists for animal models of relapse (e.g., animal models for the conditioned positive and conditioned negative reinforcement associated with dependence) has only just begun (Koob, 1995). Limited studies suggest that dopamine antagonists can block the reinstatement induced by other drugs of abuse in the intravenous selfadministration reinstatement model. For the negative reinforcement associated with drug withdrawal, there is evidence that clonidine can block conditioned withdrawal from opiates (Kosten, 1994) and chlordiazepoxide can block conditioned withdrawal from ethanol (Baldo et al., 1995). Much more work is needed in this area, particularly in developing better mod-
els and identifying mechanisms at the systems, cellular, and molecular levels of analysis.
An alternative approach to the treatment of drug dependence is the use of pharmacotherapies to alleviate the signs and symptoms of abstinence and, thus, alleviate at least part of the motivational state driving the dependence. One model for this approach that has met with significant clinical success is methadone detoxification and methadone maintenance. Early animal studies identified methadone as an orally active, long-acting opioid agonist that could block and prevent opiate withdrawal (Bigelow and Preston, 1995). An even longer-acting opiate agonist levo-alpha-acetylmethadol (LAAM) has long been under clinical investigation and is now approved by the Food and Drug Administration (FDA) for the treatment of opiate dependence (Bigelow and Preston, 1995). Nonopioid drugs, developed preclinically which also block some of the signs and symptoms of opiate withdrawal, include alpha-2-noradrenergic agonists such as clonidine. Little success has been reported, however, in preclinical attempts to block the withdrawal associated with cocaine in either animals or humans, largely because the withdrawal models have been limited (Markou and Koob, 1991); thus, development of a better model of withdrawal is also critical for progress in this area. There is some evidence that dopamine agonists can attenuate cocaine withdrawal, but the dopamine receptor subtype involved is unknown. Given the limited success of bromocriptine in the clinic, D-1 agonists, partial agonists, or even less selective dopamine agonists should be explored. Recent evidence suggests that D1 agonists are more effective than D-2 agonists in blunting the reinstatement of cocaine self-administration in animals subjected to extinction (Self et al., 1996). Ethanol withdrawal can be effectively blocked by benzodiazepines, and they continue to be the treatment of choice for detoxification (O'Brien et al., 1995). Nicotine withdrawal can be effectively eliminated by chronic, slow-release forms of nicotine delivery, an approach that forms the basis for the nicotine patch, nicotine gum, and nicotine spray in humans (Russell, 1991; Fiore et al., 1992).
Until recently, the contribution of regional brain function and neurotransmitter systems to the causes and consequences of drug abuse and other brain diseases could be addressed only indirectly through measurement of blood and cerebrospinal fluid neurotransmitter metabolites, drug challenges, and gross neurophysiological measures such as the electroen-
cephalogram (EEG). In the past decade, however, technological advances in the field of functional brain imaging have presented an opportunity to bridge the gap between basic neuroscience and clinical research. Positronemission tomography (PET), single-photon emission computed tomography (SPECT), and more recently, functional magnetic resonance imaging (fMRI) are being used in studies of the mechanisms of action of abusable drugs and of the metabolic and neurochemical changes in the brain associated with dependence.
PET and SPECT employ instruments that measure the spatial distribution and movement of radioisotopes in tissues of living subjects (Mullani and Volkow, 1992; Rogers and Ackermann, 1992). Functional magnetic resonance imaging is one of the most recent and exciting advances in brain imaging, and with PET, blood flow scans can be used to infer the activity of focal brain regions by measuring changes in blood flow by several techniques (Kaufman et al., 1996).
The fMRI procedure offers the advanced spatial resolution of MRI combined with great temporal resolution, since repeated images taken over seconds or minutes reveal discrete brain regions serially and specifically affected (e.g., during the performance of cognitive tasks or exposure to a psychotropic drug). Also, because it does not involve the use of a radioisotope, fMRI can be repeated readily. The repetition allows for measurement of changes in brain activity in response to a task or drug and how such changes may differ between normal individuals and those with neuropsychiatric disorders. Additionally, fMRI equipment is more widely available than PET or SPECT cameras. However, interpreting the evidence may not be straightforward, and a major limitation of fMRI is uncertainty as to what its signal actually reflects with respect to brain function.
There is no doubt that studies of the neurochemical state of the brains of neuropsychiatric patients made possible by PET and SPECT imaging will one day provide novel and essential information on neuropsychiatric disorders. Additional brain imaging methodologies, notably magnetic resonance spectroscopy (MRS), also promise to provide anatomical and neurochemical information that has until very recently been completely inaccessible in neuropsychiatric patients. Some of the accomplishments of these advances follow.
The availability of the short-lived positron emitter carbon-11 has made it possible to label drugs of abuse, so that PET can then be used to measure their pharmacokinetics in the human brain. The labeled drug and whole-body PET also can be used to determine the target organs for the drug and its labeled metabolites and, thus, to provide information on potential toxic effects as well as tissue half-lives. They also allow the
evaluation of the relation between the kinetics of an abused drug in the brain and the temporal relation to its behavioral effects.
Different labeled tracers can be used to assess the effects of drugs on brain function and neurochemistry, including metabolism and cerebral blood flow (CBF), neurotransmitter activity, transporter or receptor occupancy, and enzyme activity. The most widely utilized approach has been to assess the effects of acute drug administration on brain glucose metabolism and on CBF. This allows analysis of the brain regions that are most sensitive to the effects of the drug, and because the studies are done in awake human subjects, it allows analysis of the relationship between functional changes and behavioral changes in addicted and nonaddicted subjects. This strategy has been used to investigate the effects on brain glucose metabolism and/or CBF for most of the drugs of abuse.
The measurement of brain glucose metabolism with 18FDG (18fluoroD-glucose) provides an index of brain activity that is not confounded by CBF changes and hence is useful in the assessment of changes in brain function that may occur during withdrawal. For example, studies in cocaine abusers done at different times after cocaine discontinuation have shown that regional glucose metabolism changes as a function of the withdrawal phase at which the studies are performed. Cocaine abusers, and polydrug abusers, tested within one week of their last cocaine use showed significantly higher metabolic activity in frontal brain regions and in basal ganglia than normal controls (Volkow et al., 1991). In contrast, cocaine abusers tested one to four months after cocaine discontinuation showed marked reduction in frontal metabolism (Figure 3.2) (Volkow et al., 1993).
Specific receptor radioligands are useful in assessing the extent to which a particular neurotransmitter system is affected in addicted subjects. For example, in cocaine dependence (where a dysfunction in brain dopamine activity has been postulated to underlie dependence), imaging studies have documented decrements in dopamine D-2 receptor ligand binding during early and protracted cocaine withdrawal (Figure 3.3) (Volkow et al., 1993), as well as decrements in dopamine metabolism (Baxter et al., 1988). Multiple tracer studies that measure glucose metabolism and/or CBF in conjunction with specific dopamine tracers (i.e., receptors and/or transporters) permit researchers to assess the functional significance of changes in these dopamine elements. Such studies have been done to investigate the relation between brain glucose metabolism and dopamine D-2 receptors in cocaine abusers. A significant correlation was reported between dopamine D-2 receptors and glucose metabolism in orbitofrontal cortex, cingulate gyrus, and superior frontal cortex (Volkow et al., 1993). Lower values of dopamine D-2 receptor concentration were associated with lower metabolism in these brain regions.
Pain and Analgesia
Some drugs that have high abuse liability, most notably the opioid analgesics, have essential medical uses. Since its founding, NIDA has been the major supporter of research into brain mechanisms of pain and analgesia, analgesic tolerance, and analgesic pharmacology. The resulting discoveries have led to an understanding of the brain circuits that are required to generate pain and pain relief, have revolutionized the treatment of postoperative and cancer pain (Foley and Inturrisi, 1986), and have led to improved treatments for many chronic pain conditions. The major accomplishments of drug abuse research that have a significant impact on managing and relieving pain are described below.
Clinical Pharmacology of Opioids
The investigation of the potency, metabolism, analgesic effects, and side effects of opioid drugs has been a major research target of the drug abuse field since its inception. National Institute on Drug Abuse (NIDA)supported research has revealed the range of plasma opioid concentrations required for effective pain relief. Based on this research, clinicians have developed new methods of drug administration that maintain optimal levels of analgesic, including sustained-release tablets, transdermal patches, continuous drug infusions, and patient-controlled analgesic pumps. Those methods have now made it possible to keep more than 90% of cancer patients relatively comfortable for their entire course, and to eliminate most of the pain following major surgical procedures (Carr et al., 1992; Jacox et al., 1994).
Research into the Neural Circuitry Underlying Pain and Analgesia
NIDA-supported studies of the mechanism of opioid analgesia have also led to the discovery of endogenous pain-relieving circuits in the brain and spinal cord. Opioids activate analgesic areas in the brainstem, causing descending axons to release pain-inhibiting neurotransmitters in the spinal cord, blocking the entry of pain signals into the central nervous system. The finding that those inhibitory circuits use the neurotransmitters norepinephrine, serotonin, and enkephalins has led to the development of new treatments for acute and chronic pain (Wall and Melzack, 1994), including spinal administration of opioids for surgical and cancer pain (Yaksh and Malmberg, 1994) and the tricyclic antidepressants for pain caused by nerve injury (Max et al., 1992).
Molecular Biology of Pain and Analgesia
Drug abuse research has begun to elucidate the changes in gene regulation caused by acute and chronic pain and its treatment by opioids and other analgesics in animals (Hunt et al., 1987; Draisci et al., 1991). Those results provide direct applications to pain research and treatment. The amount of expression of immediate-early genes such as c-fos correlates well with the amount of tissue injury and pain behavior, offering another type of measure of pain, especially applicable to experiments in which it may be difficult to monitor behavior (Abbadie and Besson, 1994). The availability of cloned receptors from neural components mediating pain and analgesia, as well as the elucidation of second and third messenger systems provides new targets for the design of analgesic agents. In addition, the knowledge of the effects of opioids, N-methyl-D-aspartate (NMDA) antagonists, and other commonly abused drugs on genes involved in acute and chronic pain (Gogas et al., 1991) may offer insights into their effects in drug dependence and withdrawal, which like pain can have extremely aversive states.
GAPS AND NEEDS
A wealth of information has been gained concerning the actions of drugs of abuse on the brain. However, the field of drug abuse research has not, until relatively recently, taken full advantage of the revolutionary advances in molecular and cell biology and basic neuroscience that have occurred over the past two decades. New developments in molecular and cell biology open new possibilities for more basic understanding of drug abuse.
Basic Research at the Molecular Level
There are several gaps in current knowledge of drug dependence at the molecular level. One area includes genes that contribute to individual responsiveness to drugs of abuse. This includes genes encoding proteins that affect an individual's acute and chronic responses to drug exposure. A major deficiency in the field has been the choice of genes targeted for study. Most studies have focused on genes that control levels of neurotransmitters or receptors; in contrast, relatively little attention has been given to the host of genes involved in controlling intra- and intercellular signaling. Identification of genes that confer vulnerability for drug dependence would be expected to lead to the development of novel pharmacotherapies for addictive disorders.
Similarly, although progress is being made in identifying adaptations
that occur in specific brain regions in response to long-term exposure to drugs of abuse, more work is needed in this area. Identifying these adaptations will provide a more complete understanding of the ways in which drugs alter neuronal function and lead to the many long-term effects of drugs on the brain. In addition to elaborating the pathophysiology of drug dependence, a more complete knowledge of drug-induced adaptations in the brain will facilitate medication development efforts. In identifying these adaptations, an expansion of our knowledge of proteins, and of cellular and molecular processes of drug actions is needed.
Finally, only recently has there been any hint of the mechanisms by which chronic drug exposure induces adaptations in specific target proteins. The major challenge in the future will be to study many types of transcription factors and other nuclear proteins for their potential regulation by drugs of abuse, and then to relate changes in a specific transcription factor to changes in specific target proteins. In addition, increased attention should be given to posttranscriptional mechanisms, because we know that levels of a particular gene product can be influenced at the level of RNA splicing and transport to the cytoplasm, stability of the mRNA, rate of translation of the mRNA, and stability of the encoded protein. Each of these mechanisms represents a potential target for drug action.
Drug abuse research should use the potent new methods of molecular and cellular biology and the neurosciences to pay particular attention to the host of genes that control intra- and intercellular signaling following exposure to drugs of abuse, including effects on second-, third-, and fourth-messenger cascades; changes in levels of transcription factors and posttranscriptional processing; and further adaptations in target proteins.
Basic Research at the Cellular Level
Although a significant amount is known about the acute actions of opiates in certain neuronal cell types, there is a relative paucity of similar information available with respect to other drugs of abuse. For example, we still know very little about the ionic basis of the currents elicited by most dopamine receptors in the brain. A major focus of future research, then, is to utilize the most sophisticated electrophysiological methodologies available, such as patch clamping, to delineate the ionic basis of acute drug actions on the brain and the postreceptor signaling pathways through which the drugs produce these effects.
There is an even greater need to study the chronic consequences of drug exposure on the activity of target neuronal populations. Most is known about the effects of chronic stimulant exposure on activity of the mesolimbic dopamine system and its post-synaptic targets, implicated in
the motivational aspects of cocaine and other drug addictions (see above). This work has had an important impact on evolving molecular and systems analyses of drug dependence, and continued efforts are needed. Moreover, very little is known about the long-term effects of other drugs of abuse. For example, whereas considerable information is available concerning chronic opiate action on certain neuronal cell types, virtually no information is available concerning the long-term effects of opiates on neurons in the mesolimbic dopamine system and its connections. Even less is known about the consequences of chronic cannabinoid, nicotine, and psychotomimetic exposure. This type of information is critical to understand molecular phenomena within a functional context and to understand interactions among neurons at the systems level. Translation of molecular events to cellular interactions is essential to link drugs, which are molecular entities, to behavior.
Sophisticated electrophysiological methodologies should be used to delineate the ionic basis of acute and chronic effects of drugs on a variety of neuronal populations, including the linkage between changes in effects at ion channels and postreceptor signaling pathways. Studies of molecular and cellular mechanisms in brain tissue of animals with chronic drug dependence, prolonged abstinence, and relapse are of particular interest. In addition, cellular physiology, neuronal cell loss, and more subtle forms of neural injury and glial adaptations should be studied.
Basic Research at the Systems Level
A great deal has been learned in the past decade about the structure of the striatum and nucleus accumbens, the latter in particular being an important neural substrate of the acute reinforcing effects of drugs of abuse and of the motivational aspects of drug dependence. This work has delineated different subsets of neurons within these structures and has begun the arduous process of defining each subtype based on its chemical constituents (e.g., the types of proteins such as dopamine receptors and neuropeptides it expresses) and on its afferent and efferent connections. Given the important role of the nucleus accumbens in drug-related behaviors, continued efforts in this area are needed, and these efforts must be integrated more effectively with ongoing research at the molecular and cellular levels as outlined above. For example, the chemical constituents of selected subtypes of nucleus accumbens neurons represent potential targets for medication development and human genetic analyses.
In addition, the field needs to go beyond the mesolimbic dopamine system to identify other neural substrates that contribute to the complex behavioral effects of drugs of abuse. As animal models are developed that more accurately measure these aspects of dependence, the relevant
brain regions can then be identified, and these regions can be targeted for molecular and cellular analyses to understand the underlying mechanisms involved. Understanding the role of these brain structures in drug dependence will provide key information linking the well-studied mesolimbic system to other limbic structures implicated in emotions and motivated behavior and will provide a rich substrate for understanding etiology, vulnerability, and relapse.
A major goal of future research is to identify genes that contribute to individual vulnerability to drug addiction. The identification of drug addiction vulnerability genes, like the identification of any disease vulnerability gene, will require careful and thorough policy analysis and implementation. Although most research in this area has involved genetic studies in people, the focus has been on candidate genes for which there is little preclinical evidence for a role in vulnerability to dependence. For example, much of the effort in the field has focused on alleles of monoamine receptors or transporters as candidate genes. Yet, there is little if any evidence in animals or people that individual differences in the functioning of those proteins contribute to individual differences in drug responsiveness.
A promising strategy, however, which has not been employed sufficiently to date, is the use of animal models for genetic studies. This strategy is analogous to that used with success in other medical specialties. Mapping of the mouse genome, and more recently the rat genome, is proceeding at a rapid pace. By use of a variety of experimental strategies such as quantitative trait locus analysis (Belknap et al., 1993), it is now feasible to begin the process of identifying genetic loci associated with specific behavioral phenotypes related to drug dependence. It is likely that genes identified through this process will include those that encode for proteins not currently thought of as being involved in drug dependence. Identification of drug dependence vulnerability genes in animals may reveal the types of genes involved in people. Even if the same homologous genes are not involved in people, genes that encode proteins along the same biochemical pathways would be additional candidate genes for investigation. This approach would involve the targeting of far more sophisticated candidate genes for analysis, rather than a continuation of the current approach. Thus, studies of inbred rodent strains could be used to identify genes leading to different preferences for initiating or chronically maintaining self-administration of commonly abused drugs.
Such genes could then become targets for molecular and cellular genetic studies.
Transgenics and Knockouts
The advent of engineering genetic mutations in mice has been an area of explosive research interest in recent years (Capecchi, 1994; Takahashi et al., 1994). Transgenic mice refer to those in which a new, exogenous gene is expressed in the animal. Knock-out mice refer to those in which the expression of an endogenous gene has been abolished in the animal. There are also combinations of those approaches, for example, an animal in which a normal gene is removed by knock-out technology and replaced by a mutant gene with transgenic technology. It is easy to see how these genetic approaches will revolutionize the study of the normal physiological function of a given gene and its encoded protein, as well as the role of mutations in the gene in leading to various disease states (Aguzzi et al., 1994).
Research with homozygous mice, in which the gene encoding the DAT (dopamine transporter) has been disrupted, establish the central importance of the transporter as the key element controlling synaptic dopamine levels. Additionally, this research demonstrates the role of the transporter as an obligatory target for the behavioral and biochemical action of amphetamine and cocaine. The DAT knock-out mice provide a tool for the study and development of drugs used in management of dopaminergic dysfunction. These mice may also aid in determining the role of dopaminergic neurotransmission in complex behavioral paradigms such as reward, addiction, and tolerance of drugs of abuse (Giros et al., 1996).
However, the use of those genetic techniques in the neurosciences, while possessing great promise, is hindered by serious limitations. The genetic mutation is present from very early stages of development, and can lead to several layers of adaptive processes to compensate for the mutation. This is particularly problematic for the brain, where these compensations may involve altered development of synaptic connections between various neuronal cell types and even aberrant development of entire brain regions. This makes it difficult to study the physiological function of a protein (targeted by the original mutation) in the adult state. One important area for future research is to validate behavioral models of drug dependence in mice as opposed to rats. Another important need is to identify mouse strains that are useful for the generation of gene knockouts, but in which behavior has been characterized and can be reliably studied.
With these caveats, research on transgenic and knock-out animals is underway at an increasing rate. Once genetic mutant mice are generated, the next step is to identify phenotypic abnormalities. The field of drug abuse has been and will continue to be one important component of this research, because animal models of drug dependence are among the most accurate and straightforward to interpret with respect to clinical and physiological phenomena.
Signal Transduction Pathways
Progress is being made in identifying adaptations in signal transduction proteins that occur in specific brain regions following chronic exposure to drugs of abuse. One major challenge for future research is to identify and investigate a broader range of molecular and cellular targets of drugs of abuse than those currently under scrutiny. A second major challenge is to relate specific molecular and cellular adaptations to specific behavioral features of dependence, particularly drug reinforcement and motivational aspects of dependence. This will first require the development of rodent animal models that more accurately reflect the phenomenon of drug craving, which is a core clinical feature of addictive disorders. These animal models can then be used to study the functional relevance of adaptations in the cellular physiology of specific neuronal cell types; adaptations in specific signaling and structural proteins within these neurons; and ultimately, specific transcriptional, translational, and posttranslational mechanisms of the adaptive changes. The information gained may provide clues for the development of more effective medications and may aid in genetics research.
As described above, one of the major gaps in the neurobiology of drug dependence research is the integration of clinical phenomena with basic research. One area that needs attention is the further validation of current animal models (see Chapter 2) and, perhaps more importantly, the anchoring of the basic neurobiology in such models. Much of the current research focuses on the acute or semiacute administration of drugs, no validation of functional dependence (behavioral or physiological measures combined with biochemistry or molecular biology) is provided. Thus, large amounts of data are gathered on the effects of drug use, but how this is related to drug abuse and dependence is unclear.
Although models of drug self-administration and drug discrimination have provided an excellent basis for behavioral research (Chapter 2), models that can reliably reproduce the more complex behaviors of depen-
dence, relapse, withdrawal, and craving are needed. The development of animal models is a high priority, because it will enable characterization of these phenomena at the molecular, cellular, and systems levels.
Additionally, primate models have the promise of advancing knowledge in the neurobiology of drug abuse research. Primates can be trained readily in more sophisticated choice tasks that eliminate the need for controlling the rate of response, motivational, and motor confounds. The use of primate models could provide neural substrates more closely linked to the human brain from a comparative physiological perspective. Primates provide much more highly developed limbic and associative cortices for studying the neurobiology of cognitive effects of drugs of abuse and the interaction of cognition and drug development (e.g., craving and relapse). Therefore, the development of nonhuman primate models is desirable because the cortical anatomy and behavioral repertoire of primates more closely resembles those of humans.
The new imaging techniques discussed above can be used to assess the distribution of drugs of abuse in the human brain and to study neural mechanisms directly in the addicted individual. Because imaging studies are done in awake human subjects, it is possible to investigate the relation between behavior and regional brain effects, as well as between drug pharmacokinetics in the brain and the temporal course of pharmacological effects. Although the studies described above reveal the power of imaging, the field has not yet taken advantage of all of its potential.
Technologies of particular promise include fMRI because of is exceptional sensitivity and MRS because of its ability to estimate directly the concentration of many chemical components of brain tissue. Magnetic resonance also offers the ability to image brain vasculature (magnetic resonance angiography, MRA) and fluid motion (diffusion-weighted imaging, DWI). Since most drugs of abuse are vasoactive and some of the clinical sequelae associated with drug abuse may be associated with brain perfusion abnormalities, both MRA and DWI might also play important roles in understanding the mechanisms and consequences of drug abuse (Kaufman et al., 1996). Such studies may, for example, allow the initiation of investigations into the mechanism(s) by which certain drugs facilitate the emergence of aggressive behaviors.
Imaging may also prove to be valuable in the evaluation of therapeutic agents for drug dependence, for example, labeling them with positron emitters to assess their pharmacokinetics and bioavailability and to characterize their binding in the human brain. Similarly, imaging could be used to assess drug combinations and their potential toxicity, the mecha-
nisms by which environmental factors (including behavioral therapy) might affect drug abuse, or the mechanisms by which genetic factors predispose to drug abuse.
Ultimately, brain imaging and neurobiological studies have a singular purpose—to better understand drug dependence and other neuropsychiatric disorders so that more effective treatments can be developed. In this scheme, studies in molecular and cellular neurobiology identify candidate neuropathic processes relevant to neuropsychiatric disorders. Such information is validated by the tools of behavioral neuroscience in animal models of the disorders. These studies in animals then direct human genetic studies aimed at identifying specific genes that contribute to the disorders in people. Identification of proteins relevant to the disorders directs brain imaging studies to examine the status of the proteins and related systems in patients' brains. Such knowledge then defines further clinical studies of the course and treatment of specific illnesses. Of course, insight evolving from the clinical work, including brain imaging, feeds back and informs ongoing preclinical studies of the underlying mechanisms involved.
Co-Occurring Psychiatric Disorders
The major psychiatric disorders associated with drug dependence are depression and personality disorders (see Chapter 5). Epidemiological data indicate that the rates of affective disorders among drug abusers are substantially higher than the expected rates of co-occurring disorders based in the general population (see Chapters 4 and 5). For example, lifetime rates of major depressive disorders range up to 50 percent in drug dependent patients compared with only 7 percent in a community sample (Rounsaville et al., 1982, 1987, 1991), and some form of drug abuse has been identified in more than 83 percent of individuals with personality disorders (Regier et al., 1990). Psychotic disorders, such as schizophrenia, represent only about 3 percent of drug abusers, but up to 50 percent of psychotic patients have addictive disorders.
The underlying neurobiology may differ for each of those disorders and for each drug of abuse. A number of neurochemical hypotheses are based largely on pharmacological interactions with these disorders or the symptoms of these disorders. For example, people with schizophrenia taking dopamine antagonists may use cocaine to relieve the antagonistinduced dysphoria, presumably because cocaine makes dopamine available to stimulate other dopamine receptors (e.g., D-1) that can also lead to euphoria. Another speculation about schizophrenia involves excitatory amino acids and PCP (phencyclidine) leading to psychotic illnesses (Javitt and Zukin, 1991). For depression, the underlying neurobiology is less
clear because both serotonergic and adrenergic medications are helpful. Dopaminergic medications have little antidepressant efficacy, however, although dopamine seems so critical for hedonic tone or at least euphoria. Direct evidence for neurobiological connections between drug dependence and psychiatric disorders remains to be elucidated and may be studied with newly developed tools (e.g., functional brain imaging).
The utility of an animal model rests in its ability to permit the study of a disease process under controlled conditions. Animal models that recapitulate the pathogenic and functional outcomes seen with HIV infection in humans can then be used to examine the influence of drugs of abuse on HIV disease progression. Direct neurotoxic effects of drugs, in addition to their effects on immunocompetence, may contribute to an enhancement of neurological sequelae of AIDS (called neuroAIDS disease) or accelerate its onset. These studies also will help determine the nature of viral neuropathogenesis to specific brain systems relevant to drug reward. That may have significant clinical outcomes related to risk reduction in terms of altered behavioral and pharmacological sensitivity to drugs of abuse in infected individuals. Thus, behavioral analysis in animal models of viral neuropathogenesis provides a unique opportunity to study the interaction between drugs of abuse and the immune system and should go far in identifying critical viral- and host-derived factors associated with increased susceptibility to the pathobiological effects of drugs of abuse and consequent synergistic neurotoxicity. Continued development of animal models of the effects of HIV infection on the brain would be useful for studying the links between AIDS and drug abuse—e.g., effects of drugs on disease progression, and the effect of HIV on brain reward systems and behaviors relevant to risk.
Neurotoxicity of Drug Dependence
There were early reports that chronic exposure to drugs of abuse led to neuronal death. Most reports proved to be spurious, however this is still a controversial area. One example of drug-induced neurotoxicity remains well established, namely the ability of certain amphetamine derivatives to kill central monoaminergic neurons. Methamphetamine and to a lesser extent amphetamine are toxic to midbrain dopamine neurons (Seiden et al., 1975), and methylenedioxymethamphetamine (MDMA, also known as Ecstasy) is toxic to midbrain serotonin neurons (Ricaurte et al., 1988).
More recently, subtler forms of neural injury have been detected in
the brain under a variety of conditions. Chronic stress, perhaps mediated by glucocorticoids, causes pruning of dendritic spines in certain hippocampal neurons (Sapolsky, 1992). Recent work raises the possibility that neural adaptation, perhaps forms of learning, may be associated with changes in the numbers of dendrites and dendritic spines (Woolley and McEwen, 1995; Yuste and Denk, 1995). Recent evidence suggests that such subtle forms of neural injury may be induced in midbrain dopamine neurons by chronic exposure to drugs of abuse (Sklair-Tavron et al., 1995). Further work is needed to better characterize these adaptations in animal models of drug dependence and eventually to extend these studies to people by using evolving brain imaging procedures. Thus, cell loss and more subtle forms of neural injury should be studied in animal models of drug dependence.
Neurobiology of Relapse After Prolonged Abstinence
There is evidence in the clinical literature for physiological changes in people with a history of drug abuse that persist for years following the last drug exposure (Jaffe, 1990). These changes have been referred to as ''prolonged abstinence" or "protracted abstinence syndrome." Individuals who have been abstinent for years can return to a place associated with past drug exposure and quickly relapse to drug abuse (O'Brien, 1976). Individuals who took years to develop a hard-core dependence can, even after years of abstinence, descend back to that hard-core addicted state far more rapidly than before. There are relatively few preclinical studies of such types of phenomena; however, one example reports that sensitization to the locomotor activating effects of stimulants can persist for several months in rats (Robinson and Berridge, 1993). Given the clinical importance of prolonged abstinence, more preclinical research on this phenomenon is needed.
One difficulty is that it is not at all clear that the same brain regions that mediate acute drug reinforcement and, perhaps, some motivational aspects of drug dependence are involved in prolonged abstinence. Such persisting adaptations may be more likely to reside in cortical, hippocampal, and amygdaloid regions as opposed to the mesolimbic dopamine system. Again the first step in this process must be the development of animal models of prolonged abstinence.
However, we may not yet have the neurobiological resolution necessary to reveal the kinds of adaptations responsible for such long-lived phenomena as prolonged abstinence. Prolonged abstinence can be considered a form of long-term memory, and very little progress indeed has been made in establishing the neurobiological basis of long-term memory in general. Long-term memory may involve changes in the numbers or
sizes of dendritic spines of certain hippocampal and cortical neurons, or changes in the numbers and even types of synaptic terminals that innervate those neurons. Although the work is very tedious, it is possible to investigate such types of adaptation once behavioral models are developed and the relevant brain regions are identified.
A Role for Immunology in Drug Treatment
Another approach to drug abuse treatment is the development of antidrug vaccination, by which an immune response is induced in the organism that would effectively remove the drug from circulation and thus block its actions in the brain. Early work showed that immunizations can be used to blunt the reinforcing effects of morphine or heroin (Bonese et al., 1974; Killian et al., 1978). Recent evidence in cocaine abuse research suggests that synthetic analogues of cocaine can be used to produce active immunization in animals against the parent compound sufficient to block its stimulant effects (Carrera et al., 1995). Unknown at this time is how long such treatments will last and how they would affect other aspects of models of dependence. Other immunotherapies now being pursued include the development of passive immunizations (e.g., monoclonal antibodies or even catalytic antibodies could be injected into a subject to prevent a drug's action) (Landry et al., 1993). Again, the efficacy, duration of action, and impact of monoclonal or catalytic antibodies on drug dependence models remain to be explored.
Research in Analgesia and Pain
Finally, research in analgesia and pain has both informed neuroscience research on drug abuse and benefited from advances in drug abuse research. Four areas in analgesia and pain research have been highlighted for future research.
Molecular Substrates of Analgesia and Tolerance
New molecular research techniques are allowing investigators to identify some of the genes and intracellular messenger systems that are activated or suppressed by pain and analgesics (Hunt et al., 1987; Draisci et al., 1991; Gogas et al., 1991; Abbadie and Besson, 1994). These new techniques will allow a new level of analysis of the action of the body's many endogenous pain-modulating systems mediated by endorphins, enkephalins, serotonin, norepinephrine, GABA, acetylcholine, and other transmitters (Fields and Liebeskind, 1994). This in turn could lead to novel treatments for pain and make possible the prevention of tolerance to and
dependence on opioids. For example, evidence from animal models suggests that excitatory amino acid neurotransmission plays a role in tolerance to analgesia, which can be reversed or prevented by coadministration with NMDA antagonists (Elliott et al., 1994). Clinical trials of NMDA antagonist-opioid combinations in humans are just beginning.
Development of Analgesics Acting at Opioid Receptors Other than the Mu Receptor
In animals, agonists at delta, kappa, and epsilon receptors provide analgesia. In humans, such drugs might have fewer side effects or abuse liability than conventional analgesics (which act predominantly at the mu receptor). Animal studies suggest that opioids acting at different receptors may produce analgesic synergism if combined (Miaskowski et al., 1992). Research to clone receptor subtypes, develop specific drugs, and investigate their basic and clinical pharmacology will promote that goal.
Functional Brain Imaging Studies of Pain and Opioid Analgesia
Although our knowledge of pain physiology has emerged largely from studies in small animals, pain and opioid analgesia are complex human phenomena. PET and MRI are beginning to provide unique maps of the involvement of higher human brain centers in pain (Casey et al., 1994; Coghill et al., 1994; Iadarola et al., 1995). These techniques could potentially identify the areas in the brain mediating opioid analgesia and the pain-related effects on emotion, movement, and the endocrine and immune systems. Imaging methods may also be invaluable for predicting the actions of novel analgesic compounds.
Treatment of Chronic Nonmalignant Pain by Opioids
There is a consensus that acute pain and chronic cancer pain should be treated with opioids (Carr et al., 1992; Jacox et al., 1994). However, there is great controversy about the benefits and risks of long-term opioid treatment of various types of nonmalignant pain conditions such as neuropathic pain, low back pain, myofascial pain, and arthritic pain (Wall and Melzack, 1994). There are almost no data on the responsiveness of each type of pain to opioids, the rate of development of analgesic tolerance and physical dependence, and the risk of true abuse and dependence. There is a particular need for data about the risks and outcome of opioid treatment of former addicts with pain, as well as patients with pain related to human immunodeficiency virus (HIV) infection.
CONCLUSION AND RECOMMENDATION
Significant progress has been made in understanding the neural substrates of drug dependence, and yet—due to the complexity of the brain and the difficulties inherent in studying the pathogenesis of any brain disease—there is still much more work to be done. Although physical withdrawal from drugs can now be managed well, all currently available treatments for the behavioral aspects of dependence remain inadequately effective for most people. By utilizing increasingly sophisticated research techniques and methods, future neurobiological studies at all levels of inquiry—molecular, cellular, and systems—will provide essential information for developing drug abuse treatment and prevention measures.
The committee recommends continued support for fundamental investigations in neuroscience on the molecular, cellular, and systems levels. Research should be supported in the following areas: developing better animal models of the motivational aspects of drug dependence (with particular emphasis on protracted abstinence and propensity to relapse); genetics research; brain imaging; the neurobiology of co-occurring psychiatric disorders and drug abuse; animal models of the effects of HIV infection on the brain; the neurotoxicity of drug dependence; immunological approaches to drug abuse treatment; and pain and analgesia.
Abbadie C, Besson J-M. 1994. Chronic treatment with aspirin or acetaminophen reduces both the development of polyarthritis and fos-like immunoreactivity in rat lumbar spinal cord. Pain 57:45-54.
Aghajanian GK. 1978. Tolerance of locus coeruleus neurons to morphine and suppression of withdrawal response by clonidine. Nature 276:186-188.
Aguzzi A, Brandner S, Sure U, Ruedi D, Isenmann S. 1994. Transgenic and knock-out mice: Models of neurological disease. Brain Pathology 4:3-20.
Akaoka A, Aston-Jones G. 1991. Opiate withdrawal-induced hyperactivity of locus coeruleus neurons is substantially mediated by augmented excitatory amino acid input. Journal of Neuroscience 11:3830-3839.
Baldo BA, Heyser CJ, Griffin P, Schulteis G, Stinus L, Koob GF. 1995. Effects of chlordiazepoxide and acamprosate on the conditioned place aversion induced by ethanol withdrawal. Neuroscience Abstracts 21:1701.
Baxter LR, Schwartz JM, Phelps ME, Mazziotta JC, Barrio J, Rawson RA, Engel J, Guze BH, Selin C, Sumida R. 1988. Localization of neurochemical effects of cocaine and other stimulants in the human brain. Journal of Clinical Psychiatry 49:23-26.
Belknap JK, Metten P, Helms ML, O'Toole LA, Angeli-Gade S, Crabbe JC, Phillips TJ. 1993. Quantitative trait lock (QTL) applications to substances of abuse: Physical dependence studies with nitrous oxide and ethanol in BXD mice. Behavior Genetics 23:213-222.
Bigelow GE, Preston KL. 1995. Opioids. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: The Fourth Generation of Progress. New York: Raven Press. Pp. 1731-1744.
Bonese KF, Wainer BH, Fitch FW, Rothberg RM, Schuster CR. 1974. Changes in heroin selfadministration by a rhesus monkey after morphine immunization. Nature 252:708-710.
Caine SB, Koob GF. 1993. Modulation of cocaine self-administration in the rat through D-3 dopamine receptors. Science 260:1814-1816.
Capecchi MR. 1994. Targeted gene replacement. Scientific American 270(3):52-59.
Carr DB, Jacox AK, Chapman CR, et al. 1992. Acute Pain Management: Operative or Medical Procedures and Trauma: Clinical Practice Guideline. AHCPR Publication No. 92-0032. Rockville, MD: U.S. Public Health Service, Agency for Health Care Policy and Research.
Carrera MRA, Ashley JA, Parsons LH, Wirsching P, Koob GF, Janda KD. 1995. Active immunization suppresses psychoactive effects of cocaine. Nature 378:727-730.
Casey KL, Minoshima S, Berger KL, Koeppe RA, Morrow TJ, Frey KA. 1994. Positron emission tomographic analysis of cerebral structures activated specifically by repetitive noxious heat stimuli. Journal of Neurophysiology 71:802-807.
Coghill RC, Talbot JD, Evans AC, Meyer E, Gjedde A, Bushnell MC, Duncan GH. 1994. Distributed processing of pain and vibration by the human brain. Journal of Neuroscience 14:4095-4108.
Cole BJ, Cador M, Stinus L, Rivier C, Rivier J, Vale W, Le Moal M. Koob GF. 1990. Critical role of the hypothalamic pituitary adrenal axis in amphetamine-induced sensitization of behavior. Life Science 47:1715-1720.
Collier HOJ. 1980. Cellular site of opiate dependence. Nature 283:625-629.
Corrigall WA, Franklin KBJ, Coen KM, Clarke PBS. 1992. The mesolimbic dopamine system is implicated in the reinforcing effects of nicotine. Psychopharmacology (Berl) 107:285-289.
Crabbe JC, Belknap JK, Buck KJ. 1994. Genetic animal models of alcohol and drug abuse. Science 264:1715-1723.
Cunningham ST, Kelley AE. 1992. Evidence for opiate-dopamine cross-sensitization in nucleus accumbens: Studies of conditioned reward. Brain Research Bulletin 29:675-680.
Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R. 1992. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258:1946-1949.
de Wit H, Stewart J. 1981. Reinstatement of cocaine-reinforced responding in the rat. Psychopharmacology 75:134-143.
Di Chiara G, North RA. 1992. Neurobiology of opiate abuse. Trends in Pharmacological Sciences 13:185-193.
Draisci G, Rajander KC, Dubner R, Bennett GJ, ladarola MJ. 1991. Up-regulation of opioid gene expression in spinal cord evoked by experimental nerve injuries and inflammation. Brain Research 560:186-192.
Elliott K, Hynansky A, Inturrisi CE. 1994. Dextromethorphan attenuates and reverses analgesic tolerance to morphine. Pain 59:361-368.
Fields HL, Liebeskind JC, eds. 1994. Pharmacological Approaches to the Treatment of Chronic Pain: New Concepts and Critical Issues. Seattle: IASP Press.
Fiore MC, Jorenby DE, Baker TB, Kenford SL. 1992. Tobacco dependence and the nicotine patch. Clinical guidelines for effective use. Journal of the American Medical Association 268(19):2687-2694.
Foley KM, Inturrisi CE, eds. 1986. Opioid Analgesics in the Management of Clinical Pain. Advances in Pain Research and Therapy, Vol. 8. New York: Raven Press.
George FR, Goldberg SR. 1989. Genetic approaches to the analysis of addiction processes. Trends in Pharmacological Sciences 10:78-83.
Giros B, Jaber M, Jones SR, Wightman RM, Caron MG. 1996. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter . Nature 379:606-612.
Goeders NE, Guerin GF. 1994. Non-contingent electric footshock facilitates the acquisition of intravenous cocaine self-administration in rats. Psychopharmacology 114:63-70.
Gogas KR, Presley RW, Levine JD, Basbaum Al. 1991. The antinociceptive action of supraspinal opioids results from an increase in descending inhibitory control: Correlation of nociceptive behavior and c-fos expression. Neuroscience 42:617-628.
Gold MS, Redmond DE, Kleber HD. 1978. Clonidine in opiate withdrawal. Lancet 11:599-602.
Grant KA, Valverius P, Hudspith M, Tabakoff B. 1990. Ethanol withdrawal seizures and the NMDA receptor complex. European Journal of Pharmacology 176:289-296.
Guitart X, Kogan JH, Berhow M, Terwilliger RZ, Aghajanian GK, Nestler EJ. 1993. Lewis and Fischer rat strains show differences in biochemical, electrophysiological, and behavioral parameters: Studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals. Brain Research 611:7-17.
Hamamura T, Fibiger HC. 1993. Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized rats. European Journal of Pharmacology 237:65-71.
Henry DJ, White FJ. 1991. Repeated cocaine administration causes persistent enhancement of Dl dopamine receptor sensitivity within the rat nucleus accumbens. Journal of Pharmacology and Experimental Therapeutics 258:882-890.
Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T, Soubrier F, Dubay C, Cartwright P, De Gouyon B, Julier C, Takahashi S, et al. 1991. Chromosomal mapping of two genetic loci associated with blood-pressure regulation in hereditary hypertensive rats. Nature 353:521-529.
Horger BA, Giles MK, Schenk S. 1992. Preexposure to amphetamine and nicotine predisposes rats to self-administer a low dose of cocaine. Psychopharmacology 107:271-276.
Hunt SP, Pini A, Evan G. 1987. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328:632-634.
Hurd YL, Brown EE, Finlay JM, Fibiger HC, Gerfem CR. 1992. Cocaine self-administration differentially alters mRNA expression of striatal peptides. Molecular Brain Research 13:165-170.
Hyman SE, Nestler EJ. 1993. The Molecular Foundations of Psychiatry. Washington, DC: American Psychiatric Press.
Iadarola MJ, Max MB, Berman KF, Byassmith MG, Coghill RC, Gracely RH, Bennett GJ. 1995. Unilateral decrease in thalamic activity observed with positron emission tomography in patients with chronic neuropathic pain. Pain 63:55-64.
Jacox A, Carr DB, Payne R, et al. 1994. Management of Cancer Pain. Clinical Practice Guideline. AHCPR Publication No. 94-0592. Rockville, MD: U.S. Public Health Service, Agency for Health Care Policy and Research.
Jaffe JH. 1990. Drug addiction and drug abuse. In: Gilman AG, Rall TW, Nies AS, Taylor P, eds. The Pharmacological Basis of Therapeutics. 8th ed. New York: Pergamon Press. Pp. 522-573.
Javitt DC, Zukin SR. 1991. Recent advances in the phencyclidine model of schizophrenia. American Journal of Psychiatry 148:1301-1308.
Kandel ER, Schwartz JH, Jessell TM. 1991. Principles of Neural Science. 3rd ed. New York: Elsevier.
Kaufman MF, Levin JM, Christensen JD, Renshaw PF. 1996. Magnetic resonance studies of substance abuse. Seminars in Clinical Neuropsychiatry 1:1-16.
Killian A, Bonese K, Rothberg RM, Wainer BH, Schuster CR. 1978. Effects of a passive immunization against morphine on heroin self-administration. Pharmacology, Biochemistry and Behavior 9:347-352.
Koob GF. 1992a. Drugs of abuse: Anatomy, pharmacology, and function of reward pathways. Trends in Pharmacological Sciences 13:177-184.
Koob GF. 1992b. Dopamine, addiction and reward. Seminars in the Neurosciences 4:139-148.
Koob GF. 1995. Animal models of drug addiction. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: Fourth Generation of Progress. New York: Raven Press. Pp. 759-772.
Koob GF, Cador M. 1993. Psychomotor stimulant sensitization: The corticotropin-releasing factor-steroid connection. Behavioural Pharmacology 4:351-354.
Koob GF, Maldonado R, Stinus L. 1992. Neural substrates of opiate withdrawal. Trends in Neurosciences 15:186-191.
Koob GF, Markou A, Weiss F, Schulteis G. 1993. Opponent process and drug dependence: Neurobiological mechanisms. Seminars in the Neurosciences 5:351-358.
Koob GF, Heinrichs SC, Menzaghi F, Pich EM, Britton KT. 1994a. Corticotrophin-releasing factor, stress and behavior . Seminars in the Neurosciences 7:221-229.
Koob GF, Rassnick S, Heinrichs S, Weiss F. 1994b. Alcohol, the reward system and dependence. In: Jansson B, Jörnvall H, Rydberg U, Terenius L, Vallee BL, eds. Toward a Molecular Basis of Alcohol Use and Abuse. Proceedings of Nobel Symposium on Alcohol. Basel: Birhauser Verlag. Pp. 103-114.
Kosten TA. 1994. Clonidine attenuates conditioned aversion produced by naloxone-precipitated opiate withdrawal. European Journal of Pharmacology 254:59-63.
Kosten TA, Miserendino MJD, Chi S, Nestler EJ. 1994. Fischer and Lewis rats strains show differential cocaine effects in conditioned place preference and behavioral sensitization but not in locomotor activity or conditioned taste aversion. Journal of Pharmacology and Experimental Therapeutics 269:137-144.
Landry DW, Zhao K, Yang GX, Glickman M, Georgiadis TM. 1993. Antibody-catalyzed degradation of cocaine. Science 259:1899-1901.
Li TK, Lumeng L. 1984. Alcohol preference and voluntary alcohol intakes of inbred rat strains and the NIH heterogeneous stock of rats. Alcoholism: Clinical and Experimental Research 8:485-486.
Li TK, Lumeng L, McBride WJ, Waller M, Murphy JM. 1986. Studies on an animal model of alcoholism. In: Braude C, Chao HM, eds. Genetic and Biological Markers in Drug Abuse and Alcoholism. Washington, DC: National Institute on Drug Abuse. Pp. 41-49.
Littleton J, Little H, Laverty R. 1992. Role of neuronal calcium channels in ethanol dependence: From cell cultures to the intact animal. Annals of the New York Academy of Sciences 654:324-334.
Maldonado R, Koob GF. 1993. Destruction of the locus coeruleus decreases physical signs of opiate withdrawal. Brain Research 605:128-138.
Maldonado R, Stinus L, Gold LH, Koob GF. 1992. Role of different brain structures in the expression of the physical morphine withdrawal syndrome. Journal of Pharmacology and Experimental Therapeutics 261:669-677.
Malin DH, Lake JR, Carter VA, Cunningham JS, Wilson OB. 1993. Naloxone precipitates abstinence syndrome in the rat. Psychopharmacology 112:339-342.
Malin DH, Lake JR, Carter VA, Cunningham JS, Hebert KM, Conrad DL, Wilson OB. 1994. The nicotine antagonist mecamylamine precipitates nicotine abstinence syndrome in the rat. Psychopharmacology 115:180-184.
Markou A, Koob GF. 1991. Post-cocaine anhedonia. An animal model of cocaine withdrawal. Neuropharmacology 4:17-26.
Matsuda L, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561-564.
Max MB, Lynch SA, Muir J, Shoaf SE, Smoller B, Dubner R. 1992. Effects of desirpamine, amitriptyline, and fluoxetine on pain in diabetic neuropathy. New England Journal of Medicine 326:1250-1256.
Miaskowski C, Sutters KA, Taiwo YO, Levine JD. 1992. Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists. Pain 49:137-144.
Mullani NA, Volkow ND. 1992. Positron emission tomography instrumentation: A review and update. American Journal of Physiological Imaging 7:121-135.
Nestler EJ. 1992. Molecular mechanisms of drug addiction. Journal of Neuroscience 12:2439-2450.
Nestler EJ. 1994. Molecular neurobiology of drug addiction. Neuropsychopharmacology 11:77-87.
Nestler EJ, Hope BT, Widnell KL. 1993. Drug addiction: A model for the molecular basis of neural plasticity. Neuron 11:995-1006.
Nestler EJ, Fitzgerald LW, Self DW. 1995. Neurobiology of substance abuse. APA Annual Review of Psychiatry 14:51-81.
O'Brien CP. 1976. Experimental analysis of conditioning factors in human narcotic addiction. Pharmacological Reviews 27:533-543.
O'Brien CP, Eckardt MJ, Linnoila VMI. 1995. Pharmacotherapy of alcoholism. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: Fourth Generation of Progress. New York: Raven Press. Pp. 1745-1755.
Olds J. 1962. Hypothalamic substrates of reward. Physiological Reviews 42:554-560.
Olds J, Milner P. 1954. Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. Journal of Comparative and Physiological Psychology 47:419-427.
Parsons LH, Koob GF, Weiss F. 1995. Serotonin dysfunction in the nucleus accumbens of rats during withdrawal after unlimited access to intravenous cocaine. Journal of Pharmacology and Experimental Therapeutics 274:1182-1191.
Piazza PV, Deminiere JM, Le Moal M, Simon H. 1989. Factors that predict individual vulnerability to amphetamine self-administration. Science 245:1511-1513.
Piazza PV, Maccari S, Deminiere JM, Le Moal M, Mormede P, Simon H. 1991. Corticosterone levels determine individual vulnerability to amphetamine self-administration. Proceedings of the National Academy of Sciences (USA) 88:2088-2092.
Pickens RW, Svikis DS. 1988. Genetic vulnerability to drug abuse. NIDA Research Monograph 89:1-8.
Rasmussen K, Aghajanian GK. 1989. Withdrawal-induced activation of locus coeruleus neurons in opiate-dependent rats: Attenuation by lesions of the nucleus paragigantocellularis. Brain Research 505:346-350.
Rasmussen K, Beitner-Johnson D, Krystal JH, Aghajanian GK, Nestler EJ. 1990. Opiate withdrawal and the rat locus coeruleus: Behavioral, electrophysiological, and biochemical correlates. Journal of Neuroscience 10:2308-2317.
Regier DA, Farmer ME, Rae DS, Locke BZ, Keith SJ, Judd LL, Goodwin FK. 1990. Comorbidity of mental disorders with alcohol and other drug abuse . Journal of the American Medical Association 264:2511-2518.
Ricaurte GA, Forno LS, Wilson MA, De Lanney LE, Molliver ME, Langston JW. 1988. (±)3,4 Methylenedioxymethamphetamine (MDMA) selectively damages central serotonergic neurons in non-human primates. Journal of the American Medical Association 260:51-55.
Robinson TE, Berridge KC. 1993. The neural basis of drug craving: An incentive-sensitization theory of addiction. Brain Research Reviews 18:247-291.
Rogers LW, Ackermann RJ. 1992. SPECT instrumentation. American Journal of Physiological Imaging 7:105-120.
Rounsaville BJ, Weissman MM, Kleber HD, Wilber CH. 1982. Heterogeneity of psychiatric diagnosis in treated opiate addicts. Archives of General Psychiatry 39:161-166.
Rounsaville BJ, Dolinsky ZS, Babor TF, Meyer R. 1987. Psychopathology as a predictor of treatment outcome in alcoholics. Archives of General Psychiatry 44:505-513.
Rounsaville BJ, Anton SF, Carroll K, Budde D, Prusoff BA, Gawin F. 1991. Psychiatric diagnoses of treatment-seeking cocaine abusers. Archives of General Psychiatry 48:43-51.
Russell MA. 1991. The future of nicotine replacement. British Journal of Addiction 86(5):653-658.
Samson HH, Harris RA. 1992. Neurobiology of alcohol abuse. Trends in Pharmacological Science 13:206-211.
Sapolsky RM. 1992. Stress, the Aging Brain and the Mechanisms of Neuron Death. Cambridge, MA: MIT Press.
Schaefer GJ, Michael RP. 1986. Changes in response rates and reinforcement thresholds for intracranial self-stimulation during morphine withdrawal. Pharmacology, Biochemistry and Behavior 25(6):1263-1269.
Schulteis G, Markou A, Gold LH, Stinus L, Koob GF. 1994. Relative sensitivity to naloxone of multiple indices of opiate withdrawal: A quantitative dose-response analysis. Journal of Pharmacology and Experimental Therapeutics 271:1391-1398.
Schulteis G, Markou A, Cole M, Koob GF. 1995. Decreased brain reward produced by ethanol withdrawal. Proceedings of the National Academy of Sciences (USA) 92:5880-5884.
Seiden LS, Fischman MW, Schuster CR. 1975. Long-term methamphetamine induced changes in brain catecholamines in tolerant rhesus monkeys. Drugs and Alcohol Dependence 1:215-219.
Self DW, Nestler EJ. 1995. Molecular mechanisms of drug reinforcement and craving. Annual Review of Neuroscience 18:463-495.
Self DW, Barnhart WJ, Lehman DA, Nestler EJ. 1996. Opposite modulation of cocaineseeking behavior by D1- and D2-like dopamine receptor agonists. Science 271:1586-1589.
Shaham Y, Stewart J. 1994. Exposure to mild stress enhances the reinforcing efficacy of intravenous heroin self-administration. Psychopharmacology 114:523-527.
Sharma SK, Klee WA, Nirenberg M. 1975. Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proceedings of the National Academy of Sciences (USA) 72:3092-3096.
Sklair-Tavron L, Shi WX, Bunney BS, Nestler EJ. 1995. Morphological evidence of changes induced in the ventral tegmental area (VTA) by chronic morphine treatment. Society of Neuroscience Abstracts 21:1059.
Sorg BA, Kalivas PW. 1993. Behavioral sensitization to stress and psychostimulants: Role of dopamine and excitatory amino acids in the mesocorticolimbic system. Seminars in the Neurosciences 5:343-350.
Spanagel R, Herz A, Shippenberg TS. 1992. Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proceedings of the National Academy of Sciences (USA) 89:2046-2050.
Stein L. 1968. Chemistry of reward and punishment. In: Efron D, ed. Psychopharmacology, A Review of Progress (1957-1967). Public Health Service Publication No. 1836. Washington, DC: U.S. Government Printing Office. Pp. 105-123.
Stewart J, de Wit H. 1987. Reinstatement of drug-taking behavior as a method of assessing incentive motivational properties of drugs. In: Bozarth MA, ed. Assessing the Reinforcing Properties of Abused Drugs. New York: Springer-Verlag. Pp. 211-227.
Tabakoff B, Hoffman PL. 1992. Alcohol: Neurobiology. In: Lownstein JH, Ruiz P, Millman RB, eds. Substance Abuse: A Comprehensive Textbook. Baltimore: Williams & Wilkins. Pp. 152-185.
Takahashi JS, Pinto LH, Vitaterna MH. 1994. Forward and reverse genetic approaches to behavior in the mouse. Science 264:1724-1733.
Taylor JR, Elsworth JD, Garcia EJ, Grant SJ, Roth RN, Redmond DE Jr. 1988. Clonidine infusions into the locus coeruleus attenuate behavioral and neurochemical changes associated with naloxone-precipitated withdrawal. Psychopharmacology 96:121-131.
Trujillo K, Akil H. 1991. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science 251:85-87.
Vezina P, Stewart J. 1990. Amphetamine administered to the ventral tegmental area but not to the nucleus accumbens sensitizes rats to systemic morphine: Lack of conditioned effects. Brain Research 516:99-106.
Volkow ND, Fowler JS, Wolf AP, Hitzemann R, Dewey S, Bendriem B, Alpert R, Hoff A. 1991. Changes in brain glucose metabolism in cocaine dependence and withdrawal. American Journal of Psychology 148:621-626.
Volkow ND, Fowler JS, Wang G-J, Hitzemann R, Logan J, Schlyer D, Dewey S, Wolf AP. 1993. Decreased dopamine D2 receptor availability is associated with reduced frontal metabolism in cocaine abusers. Synapse 14:169-177.
Volpicelli R, Davis MA, Olgin JE. 1986. Naltrexone blocks the post-shock increase of ethanol consumption. Life Science 38:841-847.
Wall PD, Melzack R. 1994. Textbook of Pain. 3rd ed. Edinburgh: Churchill-Livingstone.
Weiss F, Markou A, Lorang MT, Koob GF. 1992. Basal extraceullular dopamine levels in the nucleus accumbens are decreased during cocaine withdrawal after unlimited-access self-administration. Brain Research 493:314-318.
Wise RA. 1989. The brain and reward. In: Liebman JM, Cooper SJ, eds. The Neuropharmacological Basis of Reward. Oxford: Clarendon Press. Pp. 377-424.
Woolley CS, McEwen BS. 1995. Estradiol regulates hippocampal dendritic spine density via an N-methyl-D-aspartate receptor-dependent mechanism. Journal of Neuroscience 14:7680-7687.
Woolverton WL. 1986. Effects of a D1 and D2 dopamine antagonist on the self-administration of cocaine and piribedil by rhesus monkeys. Pharmacology, Biochemistry and Behavior 24:531-535.
Woolverton WL, Johnson KM. 1992. Neurobiology of cocaine abuse. Trends in Pharmacological Sciences 13:193-205.
Yaksh TL, Malmberg AB. 1994. Central pharmacology of nociceptive transmission. In: Wall PD, Melzack R, eds. Textbook of Pain. 3rd ed. Edinburgh: Churchill-Livingstone. Pp. 165-200.
Young AM, Goudie AJ. 1995. Adaptive processes regulating tolerance to behavioral effects of drugs. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: Fourth Generation of Progress. New York: Raven Press. Pp. 733-742.
Yuste R, Denk W. 1995. Dendritic spines are basic functional units of neuronal integration. Nature 375:682-684.