Mark O. West - US grants

The objective of this proposed research is to study cocaine's effects on dopamine-sensitive neurons that mediate the effects of cocaine on behavior. Extracellular single-unit recordings will be obtained from the striatum and nucleus accumbens of freely moving rats. Cocaine will be administered both locally, by microiontophoresis, and systemically, by intraperitoneal injection. An important advantage of these studies is that they will be performed without the contaminating effects of anesthesia or paralysis. Instead, recordings will be obtained during the behaviors produced by cocaine (e.g., locomotion). Since locomotion itself, is correlated with changes in neural activity in these structures, a 30 minute control recording following saline injection will be obtained during locomotion on a treadmill. Dose- response curves will then be constructed of neural activity during treadmill locomotion following cocaine. Videotape recordings synchronized with the computer's acquisition of neural activity will be analyzed to isolate cocaine-induced behaviors (stereotypy) that might be correlated with neural activity and thus obscure more direct actions of the drug. The tremendous advantage of microiontophoresis is that cocaine's actions on recorded neurons can be observed directly, without contributions from feedback from the motor behaviors described above, or from activity in other brain areas that project to the striatum and accumbens. In addition to studying cocaine's effects on tonic firing rate, phasic signals will be produced by several methods, including electrical activation of the neocortical and hippocampal synaptic inputs to these structures, as well as sensory-evoked responses. All these patterns of activity will be examined, 1) in terms of cocaine's effects on dopamine-mediated neural activity, 2) in dopamine-depleted animals following 6- OHDA, and 3) in animals chronically treated with cocaine. These studies should have a major impact on our understanding of brain mechanisms related to cocaine's acute and chronic behavioral effects.

Despite abundant pathological evidence indicating that the striatum, a brain structure (caudate-putamen) is an important component of the motor system, little is known about the physiology of the striatum during normal behavior. This is true for several reasons. First, the use of anesthesia in physiological experiments, while allowing progress in understanding other regions of the brain has obscured our understanding of the striatum due to its drastic reduction in neuronal activity in this system. Furthermore, anesthetics have been shown to alter measures of dopaminergic function in the striatum (as compared with awake animals). Second, unit recordings from single striatal neurons in awake animals have seldom shown the clear, consistent correlations with motor and sensory events typical of neurons in primary motor and sensory areas. Instead responses, when present, are conditional and the details regarding the necessary conditions remain unknown. Third, single unit recordings during conscious behavior have been restricted almost entirely to the primate during head restraint. The involvement of the striatum in movement could, however, encompass a broader integration of whole body sensorimotor processing. Coordinated active movement is achieved both by causing rotational and translational movement of joints (primary movement), and by stabilizing other body parts and shifting the center of gravity so that the primary movement can occur. It is possible that conditions allowing unrestrained movement and the accompanying sensory feedback may be required for full manifestation of striatal participation in such processes. If so, electrophysiological recordings in freely moving animals, may reveal previously unobserved features elucidating the unique contribution of the striatum and basal ganglia to behavior. Dr. Mark West will study the role of the behavior of the striatum in awake, intact, unanesthetized animals.

The objective of this proposal is to provide the first characterizations of the activity of single neurons receiving dopaminergic synaptic input (nucleus accumbens, striatum and medial prefrontal cortex) during intravenous self-administration of cocaine. Units will be categorized with respect to their physiological and behavioral properties in order to establish a background for interpreting neuronal responses to cocaine. Substantial evidence indicates that a critical mechanism by which psychomotor stimulants produce motor output is via facilitation of dopaminergic transmission to accumbens and striatal neurons, and that this effect on accumbens neurons also underlies the reinforcing properties of activity, cocaine dose (cumulative infusions) and motor variables influenced by the drug (e.g., locomotion, stereotypy). To evaluate whether single cells reveal any firing patterns that might uniquely represent responses to cocaine as a reinforcer, comparisons will be made between animals self-administering vs yoked controls (contingent vs noncontingent delivery). Comparisons will be made across days in all the above measures to evaluate whether any changes occur as a function of chronic self- administration of cocaine (2-5 weeks). Neuronal firing patterns will be characterized during administration of other types of reinforcers, such as self-stimulation of the medial forebrain bundle/ventral tegmental area, or food consumption. Patterns of neuronal activity showing similarities or differences across reinforcers or across brain structures could aid in understanding neuronal reward mechanisms involved in drug abuse.

The abuse of psychomotor stimulants, cocaine and amphetamine, is thought to involve the responses of postsynaptic neurons in the nucleus accumbens and striatum to facilitated dopaminergic transmission by these drugs. The behavioral manifestations of this effect appear to be differentially mediated by these two neuroanatomical locations. Most convincingly demonstrated thus far in rats, the nucleus accumbens is implicated mainly in mediating the locomotor and reinforcing effects of these drugs, and the striatum mainly in the effects of higher doses of psychomotor stimulants on stereotyped behavior (e.g., licking, repetitive head movement). This difference almost certainly involves our recent discovery that neurons in the lateral, but not medial, striatum of the rat fire in relation to sensorimotor activity of specific parts of the body. It is not yet clear how the activity of striatal and accumbens neurons might participate in mediating psychomotor stimulant-induced stereotypy and locomotion, respectively. To address this issue, we will record the responses of single neurons in these two areas to systemic injections of cocaine or amphetamine. The firing of lateral striatal neurons related specifically to head movement or licking will be characterized during these movements after saline injection (control) and after drug injection, when the drug is altering the frequency of these movements. Similarly, drug effects will be assessed on the firing rates of accumbens neurons in the context of the moss prominent motor behavioral correlate of this structure, locomotion. The relatively high degree of knowledge about the motor behavioral variables controlling the firing rate of a neuron, particularly in the case of a neuron related to a specific, measurable movement, increases the reliability of assessing drug effects on firing. Results of the new approach proposed here and pioneered in this laboratory have suggested that the transduction of psychomotor stimulant drug action into increased frequency of certain movements may involve elevated firing of striatal neurons related to those movements, which may act to bias target neurons in premotor areas to increase the likelihood of repeating those movements. Clarifying psychomotor stimulant effects on the firing of striatal neurons related to stereotyped movements can provide novel insights regarding the response of dopamine sensitive neurons to these drugs, which may pertain to their abuse. Moreover, in light of abundant evidence linking both the reinforcing and locomotor-stimulating effects of psychomotor stimulants to the nucleus accumbens, valuable information will be provided by clarifying the effects of these drugs on locomotor-related firing of accumbens neurons.

APPLICANT'S ABSTRACT: Current views of cocaine abuse that focus on the incentive properties of drugs and their associated stimuli hold promise for guiding research that may lead to effective treatment of cocaine abuse. Impressive evidence implicating a particular neural substrate in mediating the incentive properties of psychomotor stimulants and other drugs of abuse, i.e., the mesolimbic dopamine system, potentiates the likelihood of swift and important contributions of neurobiological research toward this goal. In this proposal, using the high temporal and spatial resolution of single-cell electrophysiology to elucidate neural mechanisms of cocaine self-administration, questions are formulated within the incentive motivational framework. Recordings will be obtained from both the origin and termination of the mesolimbic system, i.e., from dopamine neurons in the ventral tegmental area (VTA) and from their target neurons in the nucleus accumbens septi (NAS). Rats in which lever pressing is maintained on a fixed ratio 1 (FRl) schedule of reinforcement by intravenous infusion of cocaine (0.7 mg/kg) exhibit regular inter-infusion intervals (mean = 6.7 min). Each reinforced lever press (RLP) is followed by a prolonged period of focused stereotypy that gradually yields to an increasing percentage of time spent in locomotion. Eventually, one or more approaches toward the lever, characterized as drug-seeking behaviors, culminate in another RLP. Single-unit recordings from mesolimbic target neurons in the NAS show cyclic patterns of firing synchronized to this behavioral cycle. While certain firing patterns conform to interpretations based on pharmacokinetics and/or on relationships of firing to motor behavior, other specific patterns do not. For example, in many cases transitions from maximum to minimum firing rate (or vise versa) are complete within < 2 sec of the RLP, in advance of any significant infusion-related increase in cocaine levels in mesolimbic areas (explicitly addressed in Specific Aim 2 by introducing on selected trials a brief delay between the RLP and the infusion). These rapid transitions in firing could reflect the cessation of drug-seeking behavior upon execution of the RLP. Supporting this interpretation are results of a preliminary experiment (the basis of Specific Aim 1), using a multiple schedule, in which a sharp elevation in firing immediately preceded the RLP during FR1 responding. Next, when the temporal pattern of cocaine infusions from the FR1 phase was presented non-contingently, infusions were no longer immediately preceded by 1) approach to and depression of the lever or 2) the sharply elevated firing; both returned when the FR1 schedule was reinstated. Thus, certain phasic NAS firing patterns do not appear to reflect only pharmacokinetics but may be related to drug- seeking behaviors, consistent with the hypothesis that mesolimbic system neurons process information regarding conditioned incentive stimuli. In Specific Aim 3, to test this hypothesis more directly than in the above studies, in which the processing of such stimuli and the timing of infusions were controlled by the animal, a programmed auditory tone will serve as a discriminative stimulus (SD) signalling the end of a variable time-out period following each RLP. After responding has been brought under stimulus control, SD-evoked alterations in firing will be assessed. In light of the considerable explanatory power of incentive- motivational theory as a framework for approaching a neurobehavioral understanding of drug abuse, the present design offers a unique opportunity to test hypotheses directly on neurons focal to this approach, i.e., the mesolimbic dopamine system.

DESCRIPTION: (Applicant's Abstract) The mesolimbic dopamine system is strongly implicated in processing the incentive properties of psychomotor stimulants and other drugs of abuse. Neurobiological studies that focus on these incentive properties of drugs and their associated stimuli may lead to effective treatment of cocaine abuse. In this proposal, using the high temporal and spatial resolution of single-cell electrophysiology to elucidate neural mechanisms of cocaine self-administration, questions are formulated within the incentive motivational framework. Recordings will be obtained from mesolimbic target neurons in the shell, core and rostral pole of the NAcc. Rats in which lever pressing is maintained on a fixed ratio 1 (FR1) schedule of reinforcement by intravenous infusion of cocaine (0.7 mg/kg) exhibit regular inter-infusion intervals (mean = 6.7 min). Each reinforced lever press is followed by a prolonged period of focused stereotypy that gradually yields to approaches toward the lever and a reinforced lever press, characterized as drug-seeking behaviors. Single-unit recordings from neurons in the NAcc show cyclic patterns of firing synchronized to this behavioral cycle. While certain firing patterns conform to interpretations based on pharmacokinetics, other patterns do not. For example, many neurons show rapid changes in firing rate within <2 sec of the reinforced lever press, in advance of any significant infusion-related increase in cocaine levels in mesolimbic areas (explicitly addressed in Specific Aim 2 by introducing on selected trials a brief delay between the reinforced lever press and the infusion). These rapid changes in firing could reflect the cessation of drug-seeking behavior upon execution of the reinforced lever press or a response to stimuli paired with the infusion. Supporting this interpretation are exciting preliminary results which indicate the feasibility of our design (the basis of Specific Aim 1): a multiple schedule in which FR1, i.e., a response contingent phase, alternates with a noncontingent phase. In the latter, the temporal pattern of cocaine infusions from the FR1 phase is presented noncontingently. Infusions are no longer immediately preceded by approach to and depression of the lever. This non-intrusive manipulation thus selectively and incisively reduces operant behavior, with pharmacokinetics held constant between the two phases. Preliminary results show that operant behavior is both necessary and sufficient for the occurrence of rapid changes in firing rate. Thus, certain phasic NAcc firing patterns do not appear to reflect pharmacokinetics but rather drug-seeking behaviors, consistent with the hypothesis that mesolimbic system neurons process information regarding conditioned incentive stimuli. In light of the explanatory power of incentive-motivational theory as a framework for approaching a neurobehavioral understanding of drug abuse, the present design offers a unique opportunity to test hypotheses directly on mesolimbic neurons which are focal to this theory.

DESCRIPTION: (Applicant's Abstract) The abuse of psychomotor stimulants such as cocaine involves the responses of postsynaptic neurons in the striatum to facilitated dopaminergic transmission by these drugs. Diverse evidence implicates specifically the lateral striatum in stereotyped behavior (e.g., licking, head bobbing in rats) induced by high doses of stimulants. The specificity of anatomical locus for this drug effect almost certainly involves our recent discovery that neurons in the lateral, but not medial, striatum of the rat fire in relation to sensorimotor activity of specific body parts. These include large populations of neurons related specifically to head movement or to oral behavior such as licking. However, it is unknown how the activity of these neurons participates in mediating stimulant-induced head bobbing and oral stereotypy, respectively. We have recently begun to address this issue by recording the firing of lateral striatal neurons related to head movement or licking during these movements after saline injection (control) and after stimulant drug injection, while the drug was influencing these movements. A remarkably consistent finding in three separate studies was that the drug effect on firing was firing rate-dependent. That is, after injection, firing rates were elevated during movements normally associated with low firing rates, but were less elevated during movements normally associated with high firing rates (of the same neuron). At high doses, normally high firing rates were strongly suppressed. Thus, even while the drug elevated low rates, inducing movements, it severely restricted the range of striatal firing rates that could be expressed in the presence of the high dose. This may account for the well documented restriction of behavioral diversity in motor behavior (i.e., focused stereotypy) induced by high doses of stimulant drugs. In Specific Aim 1, we will conduct a follow-up experiment to provide an important control for the effect of time, because the above drug effects were always obtained in the second hour, after a saline control had been obtained in the first hour. A group of animals will receive saline in the first hour and saline in the second hour. This is necessary (even though the above effects were dose-dependent) to show that the effects were attributable to the drug and not the passage of time. In Specific Aim 2, we propose to examine the effects of repeated exposure to cocaine. We will assess, as above, drug effects on firing within-session, but also assess whether those effects change across repeated daily sessions. Any changes in drug effects on firing will be examined in relation to changes in the detailed measures we obtain of motor behaviors with which firing is correlated, i.e., head movement or licking. Clarifying stimulant effects on the firing of striatal neurons related to stereotyped movements, and any changes after repeated drug exposure that may accompany sensitization of those movements, can provide novel insights regarding the response of dopamine sensitive neurons to these drugs, which may pertain to their abuse.

DESCRIPTION (provided by applicant): Neurobiological studies that focus on the incentive properties of abused drugs and their associated stimuli may lead to strategies for preventing compulsive drug seeking. According to the incentive salience hypothesis, the mesolimbic dopamine system becomes sensitized to drugs and associated stimuli, attributing "incentive salience" to their neural representations. This neuroadaptation mediates the ability of drug-associated stimuli (conditioned incentives) to elicit drug seeking, even after abstinence. The hypothesis predicts that neurons mediating incentive salience will respond to stimuli that trigger drug seeking. Using the high temporal and spatial resolution of single-cell electrophysiology during cocaine self-administration in rats, we will test that fundamental prediction: target neurons of the mesolimbic dopamine system should acquire responsiveness to a discriminative stimulus, an audible tone, that signals cocaine availability, contingent upon a lever press. Single unit activity in the accumbens shell and core, primary targets of mesolimbic dopamine projections, shown to be necessary for cocaine self-administration, will be recorded from the same neurons throughout daily training. Each lever press during the tone will produce a cocaine infusion (0.35 mg/kg) and terminate the tone. Presses in the absence of the tone will have no programmed consequence. Acquisition of tone discrimination (70 tone presentations per day, 3-6 min inter-tone interval) will be measured as reaction time to the tone, and rate of lever pressing in the absence of the tone. In two animals trained thus far, the tone did become established as a conditioned incentive, indexed by gradually reduced reaction time, such that each tone elicited an abrupt initiation of movement toward the lever. Concomitantly, the tone evoked pronounced accumbens firing for the duration of individual tone-evoked movements toward the lever. Thus, certain phasic accumbens firing patterns do appear to correlate with drug-seeking behavior, possibly in a causal way. The data are not necessarily consistent with an enhancement of the neural representation of the conditioned incentive, in that the neurons did not fire before movement, in response to the tone per se, but fired during the tone evoked movement. Nonetheless, their sustained discharge throughout approach to the lever supports a separate prediction of the incentive salience hypothesis: once triggered by the conditioned incentive, the psychological process-a neural process- (possibly downstream of the neural representation of the conditioned incentive) should be sustained until the goal is reached. Thus preliminary data are consistent with the hypothesis that neurons in the mesolimbic system process information regarding conditioned incentives. Our design can test predictions directly on mesolimbic target neurons, which are focal to incentive-motivational theory, a framework with substantial explanatory power for approaching a neurobehavioral understanding of drug abuse.

[unreadable] DESCRIPTION (provided by applicant): Rewarding effects of addictive drugs become associated with cues that subsequently trigger relapse. Animal models of reinstatement combine neurobiological and behavioral approaches that advance understanding and treatment of addiction. In some models of cue-induced relapse, a discriminative stimulus (SD) is used during conditioning, whereby the SD sets the occasion for an instrumental response (lever pressing). Other models utilize a conditioned stimulus (CS), whereby the lever press initiates the CS and drug infusion. After conditioning and withdrawal, a CS can function as a conditioned reinforcer when presented contingent upon responding, i.e., after the lever press. But random presentations of the CS (non-contingently) fail to reinstate responding. In contrast, non-contingent presentations of SDs reliably reinstate drug seeking (relapse). It is important to determine whether brain areas involved in addiction and relapse process the CS differently from the SD. Therefore, an audible tone will be conditioned as a CS (CS+) in one group and as a SD (S+) in another group of rats self-administering cocaine. Responding to a tone of a different frequency (CS- or S-, respectively) will be measured on interdigitated days on which cocaine is not available, to establish stimulus discrimination. After two weeks' abstinence, the tones will be presented in tests of reinstatement (without infusions). We will record single neuron activity in the accumbens core and shell subregions and their targets in ventral pallidum (VPdl and VPvm, respectively) during these cued reinstatement tests. In half the CS group, the CS+ tone will be presented contingently, after the instrumental response (lever press), to verify its ability to function as a conditioned reinforcer; given that core circuitry has been implicated in conditioned reinforcement and has somatomotor connections, it is predicted that core and VPdl neurons will show significant phasic activity related to the lever press during responding maintained by the conditioned reinforcer. In the SD group and the other half of the CS group, the tone will be presented non-contingently; it is predicted that 1) the S+ will but the CS+ will not increase lever pressing, and 2) shell/VPvm neurons, which mediate the ability of Pavlovian stimuli to activate instrumental responses, in the SD group will be more responsive to the S+ than those in the CS group to the CS+. Findings will help clarify neurobiological mechanisms of cue-induced relapse to drug seeking, toward developing more effective treatment strategies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): While animals and humans reliably self-administer drugs of abuse, the motivations underlying drug use are not understood. Human cocaine users retrospectively self-report both positive and negative affective states as potent precipitating factors for returned drug use. In laboratory animals it has been difficult to ascertain affective state during cocaine self-administration. For example, a rat that has experienced abstinence after cocaine self-administration might be responding to relieve withdrawal surrounding a negative affective state, or to obtain the positively reinforcing effects of the drug. Over the last fifteen years, the hypothesis that the affective states of rodents can be reliably indexed through ultrasonic vocalizations (USVs) has received much support;50-kHz USVs and 22-kHz USVs are consistently correlated with indices of positive and negative affective states, respectively, in adult rats in a variety of experimental paradigms. However, rodent USVs have not been reported during drug-seeking behavior. In order to assess affective state using an animal model of drug-seeking and relapse, we will record USVs of adult rats during cocaine self-administration and reinstatement behavior. Detailed video analyses will be utilized to determine the behaviors occurring during USV production. Furthermore, infusion of dopamine or acetylcholine into nucleus accumbens (NAcc) shell or core, respectively, unconditionally elicits USVs in rats, implicating a key structure in drug abuse in the production of USVs. Therefore, we will record NAcc core and medial shell neurons during self- administration, withdrawal, and reinstatement behavior, concurrently with USV recordings. Preliminary results of USV recordings during cocaine self-administration revealed a high prevalence of 22-kHz calls typically associated with negative affect, perhaps providing novel support for the hypothesis that one mechanism by which cocaine-seeking behavior occurs is through relief of negative affect by negative reinforcement. However, because our preliminary results were derived from a cocaine self-administration schedule of reinforcement that prevented drug 'satiety', we will also examine differences in USV production using a higher dose of cocaine that allows rats to attain drug satiety, to test whether this reduces the proportion of 22-kHz calls or increases the proportion of 50- kHz calls. These data may provide novel insight into the role of affect in various drug-seeking behaviors as well as the potential involvement of NAcc subregions during these processes. PUBLIC HEALTH RELEVANCE: Complex affective and motivational factors are involved in substance abuse. Animal models provide substantial insight into drug abuse, in terms of behavioral, pharmacological, neurochemical, neuroanatomical and neurophysiological measures obtained. Animal drug abuse literature lacks a description of animal ultrasonic vocalizations (USVs), which have been demonstrated to signal different affective states. Virtually all the above measures obtained in animal models of drug abuse could be enhanced if they were coupled to corresponding USVs signaling affective state. Indeed, USV production is linked primarily to a neural structure heavily implicated in drug abuse: the nucleus accumbens. Proposed experiments will explore the relationships of USVs and accumbens firing patterns during cocaine self-administration, withdrawal, tests of cocaine cue reactivity, and relapse. Results could enlighten interpretations 1) of what animals may be processing during what are considered "drug seeking" behaviors, and 2) of potential affective correlates of neurophysiological activity.

DESCRIPTION (provided by applicant): Research into the neural bases of cocaine abuse has established an essential role of the mesolimbic dopamine (DA) system innervating the nucleus accumbens (NAc). Recently, the nigrostriatal DA system innervating the dorsolateral striatum (DLS), a region necessary for acquiring habits, has been implicated in cocaine abuse. According to this view, drug self- administration gradually recruits striatal circuitry beyond the ventromedial striatum (accumbens), eventually spreading to the DLS. Rats will self-administer cocaine in a tone discrimination task, 6 hours/day, 7 days a week for 4 weeks to model both the long access/escalation pattern of intake described by Ahmed and Koob (1998) and the accompanying changes in striatum that have been shown to occur in the chronically drug-exposed brain by Porrino et al. (2004). Among numerous single unit recording studies during cocaine self-administration in well trained subjects, none have ever i) recorded DLS neurons, ii) recorded NAc shell, core and DLS simultaneously, or iii) tracked their phasic firing patterns from the first session throughout an extended period of cocaine self-administration. All three are the focus of the proposed studies. The instrumental response will be a vertical head movement or licking a dry spout, in order to target DLS neurons related specifically to one of those two movements. Single unit activity will be recorded simultaneously from NAc medial shell and core neurons, from non-sensorimotor DLS neurons and from DLS neurons related specifically to vertical head movement or to licking. Single neurons will be tracked across sessions to assess changes within and between subregions. The spatial and temporal resolution of the proposed single unit recordings would bring an unprecedented level of detail to the tracking of long-term neural changes associated with chronic cocaine abuse, and assure the acquisition of new and valuable information that will be necessary ultimately for mechanistic explanations. PUBLIC HEALTH RELEVANCE: Chronic cocaine self-administration causes changes in circuitry of the human basal ganglia. Rats will self-administer cocaine in a long access/escalation pattern to model these reported changes. Single neuron impulse activity will be tracked across self-administration sessions to assess changes in rat basal ganglia circuits. The spatial and temporal resolution of single unit recordings would provide valuable information relevant to mechanistic explanations of long-term neural changes associated with chronic cocaine abuse.