Nancy R. Zahniser - US grants

Cocaine blocks neuronal dopamine (DA) uptake, prolongs the presence of DA in the synapse and thus acts indirectly as a DA receptor agonist. The positive reinforcing effects of cocaine have been hypothesized to involve these actions upon mesolimbic and mesocortical dopaminergic systems. The phenomenon of cocaine-induced behavioral sensitization, which is induced by repetitive cocaine administration, is associated with mesolimbic and nigrostriatal dopaminergic systems. These observations have aroused considerable interest in understanding changes in DA sensitivity produced by cocaine in these pathways. The hypothesis to be tested is that dopaminergic systems become sensitized with single or repeated administration of cocaine because the properties of the presynaptic DA transporter protein are altered leading to uncoupling of the neuronal uptake pump from its recognition binding site and/or enhanced transport of DA out of the terminal via a reversal of the pump. Temporal changes in 3H-DA uptake, its recognition binding site (3H-mazindol binding) and amphetamine-induced release of endogenous DA in the nigrostriatal, mesolimbic and mesocortical systems will be compared to changes in locomotor activity and stereotypic behavior induced by single and repeated cocaine administration. Properties of DA receptors will also be measured to determine whether or not they are regulated in a manner to compensate for the changes in the presynaptic release/uptake mechanism. Presynaptic DA receptor function will be assessed by measuring the D-2 receptor modulation of electrically-evoked release of endogenous DA from slices. The properties of 3H-mazindol binding sites and D-2 and D-1 DA receptors will be determined using digital subtraction autoradiography in tissue sections containing either cell bodies or terminals of these three dopaminergic systems. Pharmacological controls for these experiments will investigate whether administration of drugs that block catecholamine uptake, act as direct DA agonists or are local anesthetics lead to similar behavioral sensitization and neurochemical changes. This set of experiments will test the specificity of the observations made following cocaine administration. Recently, desipramine and bromocriptine have been reported to be successful in the treatment of cocaine abuse in humans. Whether current or subsequent administration of these agents, together with cocaine, reverses any of the cocaine-induced changes in behavioral sensitization and/or DA neurochemistry will be determined. These experiments should help to resolve whether alterations in the presynaptic DA transporter are responsible for the behavioral sensitization produced by cocaine and whether these neurochemical changes can be reversed by pharmacological treatment.

Dopamine (DA) inhibits the evoked release of the neurotransmitters DA, acetylcholine (ACh) and gamma-aminobutyric acid (GABA) in rat striatal. The unifying hypothesis of this proposal is that these striatal release-modulating receptors are all of the D-2 subtype but that their second messengers and regulation may differ. Differences in the pharmacological profiles of these three receptors have been reported. These may reflect differences in (1) the neuronal location of the receptors, (2) the amounts of synaptic DA to which the receptors are exposed and (3) the contributions from D-1 DA receptor activation. The pharmacological profiles will be determined from dose-response curves for DA receptor agonist and antagonist modulation of electrically-evoked 3H-DA, 14C-ACH and 3H-GABA release from rat striatal slices. Tetrodotoxin will be used to determine whether axonal transmission is necessary for the action of these receptors. Synaptic DA levels will be depleted with alpha-methyl-p-tyrosine. The influence of concomitant D-1 DA receptor activation will be evaluated using Schild analysis. Next, the involvement of multiple mechanisms - including coupling to GTP binding proteins, inhibition of adenylate cyclase activity, decreased activation of voltage-sensitive calcium channels and activation of a potassium conductance - in striatal D-2 receptor modulation of neurotransmitter release will be investigated. Specifically, the effects of (1) treatment with pertussis toxin, (2) agents that elevate cAMP and calcium levels and (3) calcium and potassium channels blockers on the actions of these striatal D-2 release modulatory receptors will be investigated. Lastly, it is likely that the D-2 receptors that modulate evoked 3H-DA release, because of their inhibitory presynaptic nature, will be regulated differently from those that modulate 14C-ACh and 3H-GABA release. Changes in receptor sensitivity will be induced by chronic administration of two DA receptor antagonists or by nigrostriatal denervation. In order to determine whether changes in the sensitivity of these release-modulating receptors are those measured by radioligand binding assays, the changes in the release- modulating receptors will be compared with those in the DA receptors measured with quantitative autoradiographic receptor analysis. Striatal D-2 DA receptors appear to be important both in the therapeutic actions of antiparkinsonian agents and in the untoward side effects of antipsychotic agents. It is our hope that the results of the proposed experiments will help to distinguish both pharmacologically, as well as physiologically, the specific subpopulation of release-modulating D-2 DA receptors in the striatum.

The proposed experiments will test the hypothesis that both enhanced dopamine (DA) overflow and increased functionality of D-1DA receptors contribute to the behavioral sensitization observed following repeated cocaine administration. Specifically, we hypothesize that following withdrawal from repeated cocaine administration, evoked DA overflow from nerve terminals will be increased only when a cocaine challenge is given but that terminal release-modulating D-2DA autoreceptors and postsynaptic D-1DA receptors will be supersensitive whether or not a cocaine challenge is given. In vivo voltammetry in anesthetized rats and n vitro slice preparations will be used to study cocaine-induced changes in DA overflow in the nucleus accumbens (NAc) and striatum. Rats will be treated with saline or cocaine (10 mg/kg, i.p.; twice daily) for 14 days and withdrawn for 7 days. Using in vivo voltammetry, electrical-and K+-evoked DA overflow will be compared in the NAc and striatum following no cocaine challenge, local cocaine or DA challenge and systemic cocaine challenge. These experiments will indicate whether cocaine challenge is required to increase evoked DA overflow, whether the change in overflow is observed when the stimulus is restricted to the terminal or dependent on the cell body and/or impulse flow and whether changes in vesicular release and/or the DA transporter are involved. Slice preparations will be used to investigate changes in the ability of cocaine to enhance DA overflow and changes in the ability of terminal D-2DA autoreceptors to modulate this overflow. In vitro radioligand binding and adenylate cyclase assays will be used to investigate changes in agonist interactions with D-1 and D-2 DA receptor subtypes in these same regions. Increases in the proportion of high affinity receptors in the magnitude of the guanine nucleotide-induced shift in agonist affinity and/or receptor-coupled adenylate cyclase activity could contribute to enhanced postsynaptic DA responsiveness following repeated cocaine administration. To further test the relationship of the observed neurochemical and behavioral changes persist for the same duration. Also, pretreatment with either SCH-23390, a D-1 DA receptor antagonist, or with MK-801, an N-methyl-D-aspartate receptor antagonist, has been reported to block development of stimulant-induced behavioral sensitization. These two pharmacological pretreatments will used to block cocaine-induced behavioral sensitization and to determine the effect on the cocaine-induced neurochemical changes identified in the previous Specific Aims. The results of these studies will enhance our understanding of persistent changes in DA neurochemistry and regulation of the mesolimbic and nigrostriatal DA systems that occur as a result of repeated cocaine administration. It is our hope that understanding these changes will enhance our ability to predict the long-term consequences of low-level,intermittent use of cocaine on central DA systems.

This is a request for an ADAMHA RSDA (Level II). The proposed experiments will investigate whether enhanced dopamine (DA) overflow and D-1 DA receptor function contribute to the behavioral sensitization observed following repeated cocaine administration. Specifically, we hypothesize that following withdrawal from repeated cocaine administration, evoked DA overflow from nerve terminals will be increased only when a cocaine challenge is given but that the functionality of terminal release- modulating autoreceptors and postsynaptic D-1 DA receptors will be greater whether or not a cocaine challenge is given. In vivo voltammetry in anesthetized rats and in vitro slice preparations will be used to investigate the DA transporter and evoked DA overflow following withdrawal from repeated cocaine administration. Results in the nucleus accumbens (NAc) and striatum will be compared. Local challenge with DA or cocaine and systemic challenge with cocaine will be utilized. The results will indicate (1) whether cocaine challenge is required to increase evoked DA overflow, (2) whether the change in overflow is observed when the stimulus is restricted to the terminal or whether it is dependent on the cell body and/or impulse flow and (3) whether changes in vesicular release and/or the DA transporter are involved. In vitro binding and second messenger assays will be used to investigate changes in agonist interactions with D-1 and D- 2 DA receptor subtypes in these same brain regions. To test further the relationship of the observed neurochemical changes with behavioral sensitization, the time course and dose-dependency of the neurochemical and behavioral changes will be compared. Pretreatment with SCH-23390, a D-1 DA receptor antagonist, or with MK-801, an N-methyl-D-aspartate receptor antagonist, both of which have been reported to block stimulant-induced behavioral sensitization, will also be used to test the relationship between the neurochemical and behavioral observations. The results of these studies will enhance our understanding of persistent changes in DA neurochemistry and regulation of the mesolimbic and nigrostriatal DA systems that occur as a result of repeated cocaine administration. It is our hope that understanding these changes will enhance our ability to predict the long-term consequences of low-level, intermittent use of cocaine on central DA systems. The importance of this RSDA to my professional growth will be (1) the opportunity to focus on this cocaine research project, (2) the opportunity to learn new techniques in other scientists' labs that will enhance the scope of the proposed research and (3) the opportunity to interact with experts in this field in order to extend the breadth of my own research into new areas. The four major areas that would be assimilated into my cocaine research program as a direct consequence of an RSDA include in vivo electrochemical recording in behaving rats, serotonin systems, molecular biological approaches and pharmacogenetics.

The striatum is a major component of the basal ganglia, through which cortical input is integrated and focused to particular brain regions involved in motor planning and motor memory. Striatal activity is strongly modulated by nigrostriatal dopamine (DA) input. Both major striatal efferent pathways projecting to the globus pallidus (GP) and substantia nigra pars reticulata (SNr) use gamma-aminobutyric acid (GABA) as their neurotransmitter. Adenosine is a ubiquitous neuromodulator, but the observations that high affinity A(2a) adenosine receptors in striatum are associated exclusively with striatopallidal neurons and that D2 DA and A(2a) receptors have opposing actions in the striatum suggest that adenosine may play a unique role in the basal ganglia. The overall hypothesis being tested is that an important mechanism by which DA and adenosine regulate synaptic transmission in basal ganglia is modulation of stimulation-evoked release of DA and GABA. The primary test system to be used in these experiments will be electrically-stimulated release of endogenous DA or GABA from rat brain slices. The first set of experiments will address whether in the striatum "postsynaptic" D2 receptors that modulate the GABA release can be differentiated, particularly with respect to coupling mechanisms and/or drug-induced regulation, from presynaptic D2 autoreceptors that modulate DA release. Schild analysis, sensitivity to tetrodotoxin, coupling to G proteins and K+ channels and regulation induced by subchronic administration of neuroleptics will be determined. The second set of experiments will determine the pharmacological profile of adenosine receptor subtypes that modulate GABA release in striatum. The potential antagonism between A(2a) and D2 receptor agonists will also be explored. The third set of experiments will investigate whether there is a regional specificity in the modulation of GABA release by D1 and D2 DA receptors, as well as A1 and A2a adenosine receptors, in micropunches of GP, SNr and striatum. Initially, the importance of autoinhibition of GABA release via GABA-B receptors will be evaluated. Next, the pharmacological profiles, putative enabling effects of D1 receptors on D2 receptor- mediated activity and interactions between A2a and D2 receptors will be explored in these same brain regions. Striatal ibotenic acid lesions will be used to determine whether or not a particular modulatory action in GP or SNr is selectively attenuated following degeneration of the striatopallidal and striatonigral pathways. Additionally, whole-cell patch-clamp and conventional intracellular recording in slices containing both striatum and GP will be used to further define the locus, of the modulatory drug, actions in the striatopallidal pathway. These experiments will determine whether different DA and adenosine receptor subtypes are involved in the modulation of GABA release GP and SNr and whether the same relationship exists in striatum. Understanding the basic biology of the two major striatal efferent pathways may suggest new pharmacological strategies for independently manipulating their function, which, could prove useful in the treatment of movement disorders such as Parkinson's disease and Huntington's disease.

DESCRIPTION: (Applicant's Abstract) This application is a request for continued funding of an Independent Scientist Award (ISA; K02) to support Dr. Nancy Zahniser in the Department of Pharmacology, University of Colorado School of Medicine. The importance of this ISA to Dr. Zahniser's career and professional growth is that it will continue to (1) minimize her administrative and teaching responsibilities, (2) enhance her focus on drug abuse research, (3) allow her to devote maximal effort toward new research directions initiated during the previous support period and (4) provide increased opportunities to further extend the scope of her research. During the past four years. Dr. Zahniser's lab developed and characterized an in vivo electrochemical method to measure exogenous dopamine (DA) clearance, a measure of DA transporter (DAT) activity, in discrete brain regions of anesthetized rats. Using this approach, they demonstrated that, consistent with behavioral sensitization, the ability of cocaine to inhibit DA clearance is persistently enhanced in nucleus accumbens, but not caudate-putamen, following withdrawal from repeated cocaine administration. Critical to the experiments proposed in the present application, she and her collaborators have recently extended this methodology so that behavior and DAT activity can be measured concurrently in freely-moving rats. In a collaborative project initiated during Dr. Zahniser's sabbatical, human DAT expressed in Xenopus oocytes was shown to be electrogenic, to possess some ion channel-like properties and to be voltage-dependent. Experiments proposed here will explore the latter finding further in the oocyte expression system and in brain slices from rats and D2 DA receptor knockout mice. Specifically, the proposed experiments will test the following hypotheses: (I) that the rate at which exogenous DA is cleared from the extracellular space parallels the rate of endogenous DA clearance, (2) that persistent changes in DAT activity in specific brain regions contribute to the expression of cocaine-induced behavioral sensitization and (3) that D2 DA autoreceptor-induced changes in membrane potential transiently regulate DAT activity. The results of these studies will enhance understanding not only of persistent changes in brain DA systems that contribute to behavioral sensitization but also of mechanisms by which DAT activity is transiently regulated.

Repeated intermittent administration of cocaine results in behavioral sensitization. The overall goal of this project is to identify neurochemical alterations responsible for this persistent phenomenon. The experiments proposed in this application will test the hypotheses that persistent changes in dopamine (DA) and serotonin (5-HT) transporter function in specific brain regions, as well as persistent changes in cocaine disposition in the brain, contribute to the expression of cocaine- induced behavioral sensitization. The initial set of experiments will examine the role played by increased brain concentrations of cocaine in behavioral sensitization. Higher drug levels are achieved in the brain following repeated, as compared to acute, intraperitoneal (i.p.) administration of cocaine. This is not expected to be the case with intravenous (i.v.) administration. Brain cocaine levels will be measured following acute and repeated i.v. cocaine. Also, it will be investigated whether repeated i.v. cocaine administration produces changes similar to repeated i.p. administration in terms of behavior, DA clearance and DA transporter binding. During the past funding period, in vivo electrochemical (EC) recording has been used to characterize the disappearance of exogenous (locally-applied) DA in dorsal striatum and nucleus accumbens (NAc) of urethane-anesthetized rats. This measure of clearance reflects neuronal DA transporter function. In the second set of experiments, these studies will be extended to examine the clearance of endogenous DA, released in response to electrical stimulation, in the anesthetized rat. The aim will be to compare endogenous and exogenous DA clearance rates. The third set of experiments will examine cocaine-induced changes in clearance of stimulation-evoked endogenous DA in dorsal striatum and NAc of unanesthetized, freely behaving rats. Concurrent in vivo EC and behavioral measurements will test the hypothesis that hypersensitivity of the DA transporter, particularly in NAc, is directly related to cocaine- induced behavioral sensitization. The effects of repeated, intermittent injections, which should result in behavioral sensitization, and continuous infusion, which should result in behavioral tolerance, will be compared. The final set of experiments will examine the possible role of 5-HT in cocaine sensitization. Increases in both extracellular 5-HT levels in NAc and 5-HT transporter binding sites in medial prefrontal cortex (MPFC) have been reported in cocaine-sensitized rats. Behavioral experiments will determine whether selective lesioning of 5-HT neurons with 5,7- dihydroxytryptamine alters expression of sensitization. Exogenous 5-HT clearance rate will be characterized using in vivo EC recording in NAc and MPEC of anesthetized rats. Subsequently, it will be determined whether 5- HT clearance in these two brain regions is differentially and persistently altered following repeated cocaine administration. Elucidation of the mechanism(s) underlying behavioral sensitization in animals may enhance our ability to predict and understand the long-term consequences of cocaine abuse, including drug-induced psychosis, in humans.

DESCRIPTION (provided by applicant): This application requests continued support for a Postdoctoral Training Program, now in its 25th year, designed to produce well-trained independent researchers in the area of alcohol abuse and alcoholism. Funds are requested to support 8 fellows per year (levels 0-2), with the average duration of support being two years. This multi-disciplinary program has three broad areas of training: pharmacology, genetics, and behavior. These areas are covered by 30 well-funded training faculty, who focus both on basic and clinical alcohol/drug abuse research. The majority of the faculty currently on the grant will continue to participate. However, 13 new training faculty - some more junior and some senior in rank - have been added. A number of units within the Denver and Boulder campuses of the University of Colorado are involved: the Departments of Pharmacology, Psychiatry, Pharmaceutical Sciences, Psychology and Division of Clinical Pharmacology; the Institute for Behavioral Genetics; the NIAAA-funded Alcohol Research Center (ARC); and the NIDA-funded Center on Antisocial Drug Dependence: Genetics & Treatment. Trainees with doctoral degrees are recruited from a broad range of disciplines. Concerted efforts have been made to recruit trainees from under represented racial/ethnic groups and to contribute to longer-term programs to "fill the pipeline". Trainees work primarily in one lab, but collaborative interactions with other preceptors and trainees are strongly encouraged. All of the preceptors use state-of-the-art pharmacological, genetic and/or behavioral approaches. Training in quantitative and molecular genetics, combined with a broad range of pharmacological approaches, allows the fellows to dissect the molecular, cellular and genetic bases for behavioral reactions to drugs and the environment. Another important characteristic of this Training Program is the opportunity to participate in interactions between basic science and clinical practice. Contact with other faculty and trainees through a regular seminar series; ARC retreats; and various courses, including Ethics in Research, complete the training environment. Trainees are also encouraged to write individual NRSAs and present their work in local seminars, as well as at national and international meetings. Through this Training Program, fellows be-come familiar with behavioral pharmacogenetics as a discipline and its various approaches used in solving important problems related to alcohol actions and alcoholism. Past trainees from this program have been very successful, and a number are continuing to make significant contributions in the alcohol and drug abuse fields.

DESCRIPTION: (Provided by Applicant): This new application for a "K05 Senior Scientist Award" from NIDA requests 5 years of support for Nancy R. Zahniser, Ph.D., Professor of Pharmacology, University of Colorado School of Medicine. The Department of Pharmacology is distinguished by its strengths in neuropharmacology, alcohol and drug abuse research; strong funding record; scientific impact; and collaborative atmosphere. Dr. Zahniser has had a NIDA "K02 Independent Scientist Award" for the past 9 years, and this support has been crucial to her career, allowing her not only to focus on drug abuse research but also to expand the scope of her research. For example, her early work suggested that the dopamine transporter (DAT) contributes to persistent expression of cocaine-induced behavioral sensitization. She helped to develop new electrochemical recording technology to measure concurrently DA clearance, a measure of in vivo DAT activity, and behavior in freely-moving rats. She and her graduate student have now used this method to confirm this relationship. Thus, only rats that showed cocaine-induced changes in DAT also exhibited behavioral sensitization. Development of this technology was instrumental in her recently receiving a MERIT Award to support her work. These results have lead to her long-term goal of understanding DAT regulation in the brain. She has identified membrane potential, protein kinases, DA D2 receptors and substrates as regulating DAT activity and expression. Her results, like those from other labs, suggest that DAT regulation occurs largely via changes in cell surface expression. Thus, she initiated a collaboration with Dr. Alexander Sorkin to study the basic molecular machinery and protein-protein interactions that occur during endocytic DAT trafficking in model cell systems and neuronal preparations. This K05 Award will provide her "protected time" to focus on this new direction, as well as on new projects studying DAT expression/function in stem cells and in cocaine-sensitive and -insensitive mice, all of which will significantly contribute to her career development and the drug abuse field. One of the most important and unique aspects of her work is the desire and ability to test hypotheses about DAT, derived from model systems, back in the brain. Dr. Zahniser has also consistently contributed to the drug abuse field by reviewing grants/manuscripts, serving on advisory panels and organizing symposia/meetings. She is active in training. Six of her 14 former graduate students and postdoctoral fellows are now doing drug abuse/alcohol research as independent faculty investigators. All 5 of her current trainees are working on drug abuse/alcohol research projects.

[unreadable] DESCRIPTION (provided by applicant): The University of Colorado Health Sciences Center Pharmacology Graduate Training Program is requesting annual support for nine pre-doctoral students throughout a five-year period. The Pharmacology Training Program is one of the longest funded NIGMS Pharmacology Training Programs (twenty-five plus years). This Graduate Training Program distinguishes itself by providing an environment in which students can obtain a broad-based integrative perspective of science, training in the foundation of knowledge that defines pharmacology, and sophistication in specialized, modern, research areas. The Pharmacology Training Faculty (47 members) is drawn both from within and from outside of the Department of Pharmacology, in the School of Medicine, to provide training opportunities in the areas of pharmacogenetics, neuropharmacology and addictive processes, cellular/molecular pharmacology and oncology, molecular structure and drug design, bioinformatics and computational pharmacology, as well as clinical pharmacology. The M.D. and Ph.D. preceptors in the Program are seasoned researchers and mentors with significant extramural funding. The sources of students entering the Program include direct applicants to the Program, as well as individuals who transition from umbrella programs such as the Biomedical Sciences Program and the Medical Scientist Training Program into the Pharmacology Graduate Training Program. The Pharmacology Graduate Training Program has curricular options that include the basic pharmacology track, the molecular structure track, and the bioinformatics track. These options provide a broad array of opportunities for student career development in the pharmacological sciences. The hallmarks of our Program are a strong didactic component, requirements for laboratory rotations, a strong emphasis on student presentations in seminar settings, and a wide choice of thesis research options. The Pharmacology Research Training Program has been successful in recruiting minority applicants and matriculating all of its students within a four-to-six year period. Student presentation at national meetings, publications in peer-reviewed journals, and student competitiveness for individual NRSA awards is one measure of the successful training of our students. Additionally, the retention of the graduates of our Program in academic, industry, and government positions is another measure of the success of our Program training. With a renewal for five years of funding, the Program will continue to meet the national demands for individuals, trained as pharmacologists, who are individually astute researchers, can be multi-disciplinary research team members, and also have the breadth of knowledge to plan and communicate effectively across a spectrum of technologies. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This application for a K05 Senior Scientist Award requests an additional 5 years of support from NIDA for Nancy Zahniser, Ph.D., Professor of Pharmacology and Neuroscience, U Colorado School of Medicine. The U Colorado is distinguished by its strong drug/alcohol addiction research, funding record, training programs and collaborative atmosphere. Dr. Zahniser's support by a NIDA K05 Award for the past 4 years has been crucial in allowing her to focus on and expand the scope of her drug addiction research. Her research seeks to understand how the differential initial behavioral responsiveness of individual rats to cocaine predicts their vulnerability to addiction, how the dopamine transporter (DAT) contributes to these phenotypes and how functional DAT expression is regulated. Recently, she began a new collaboration with Dr. Richard Allen that has enhanced her lab's behavioral testing repertoire to include conditioned place preference (CPP) and, very soon, self-administration. Overall, her work has shown that the initial locomotor response to cocaine can predict the extent of cocaine-induced locomotor sensitization and CPP, suggesting that factors like DAT that contribute to the different phenotypes may help to explain individual differences in reward. Such findings have sparked her interest in understanding how DAT is regulated and her productive collaboration with Dr. Alexander Sorkin. DAT regulation occurs largely via altered trafficking and cell surface expression. Their work has resulted in novel insights about the cellular mechanisms utilized during endocytic DAT trafficking in model cell systems. They are currently extending these findings to brain preparations. The ability to test hypotheses about DAT derived in model systems, back in the brain, remains one of the most important and unique aspects of Dr. Zahniser's work. Continued K05 support will provide her protected time to focus on new research directions and mentoring, as well as to utilize her recent leadership training in organizing a new university-wide drug addiction research center, integrating basic, clinical and treatment research. Dr. Zahniser contributed/s to NIDA's mission by serving on NIDA Council and IRP Board of Scientific Counselors, external advisory boards for other addiction centers, and as a reviewer. She is active in mentoring students, postdoctoral fellows and junior faculty who are supported by NIDA. Ten of her 20 former trainees are faculty members/senior fellows who continue independently to focus on drug/alcohol addiction research. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application requests renewal of a NIDA-sponsored MERIT Award as a R01. The overall goal is to under- stand why individuals differ in cocaine-induced activation and, specifically, i) how the dopamine (DA) trans- porter (DAT) contributes to this differential responsiveness and ii) how this individual variability is predictive of cocaine abuse liability. Adult outbred male Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on their differential open-field locomotor activity following a low dose cocaine injection. These initial differences in cocaine activation are explained, in part, by how effectively cocaine inhibits the DAT in dorsal striatum (dSTR) and nucleus accumbens (NAc). This classification also predicts repeated cocaine-induced behaviors associated with reward and reinforcement, in that LCRs, more than HCRs, exhibit locomotor sensitization, conditioned place preference and motivation to self- administer cocaine. Differential LCR/HCR DAT inhibition would be expected to result in differences in extra- cellular DA levels and DA receptor (R) stimulation. Aim 1 will use i) in vivo microdialysis to define the overall relationship between endogenous extracellular DA in dSTR and NAc core and acute and repeated cocaine- induced locomotor activation in LCRs and HCRs and ii) new state-of-the-art in vivo electrochemical recording technology to assess real-time changes in cocaine-induced endogenous extracellular DA levels in dSTR and subregions of NAc (core and shell) in freely-behaving LCRs and HCRs. Aim 2 will explore potential differences between LCRs and HCRs in cocaine-induced i) rapid DAT regulation (trafficking) in dSTR and NAc and ii) longer-term DAT and DA R regulation that could help to explain the behavioral differences. These experiments will measure open-field activity, cell surface DAT levels, [3H]DA uptake kinetics, and levels of the orphan G- protein-coupled receptor GPR37. Quantitative autoradiographic analysis of in vitro radioligand binding will be used to assess regulation of DATs, D1Rs, D2Rs and agonist-stimulated G-protein coupling. Aim 3 will deter- mine how LCR/HCR classification predicts addiction-related effects of cocaine by measuring i) sensitization during acquisition of cocaine self-administration and cocaine pre-exposure, ii) sensitization to motivational effects of cocaine measured under a progressive ratio schedule of reinforcement, and iii) incentive sensiti- zation in the context of cue-elicited reinstatement of responding for cocaine and responding under a second- order schedule of reinforcement. Concurrent in vivo electrochemical recording will be used to address particular questions in the self-administration experiments. Together, the results will provide novel insights as to why initial insensitivity to cocaine locomotor activation is associated with the more addiction prone pheno- type, how both rapid and longer-term DAT adaptations and their consequences contribute to this individual variability, and how sensitization impacts cocaine self-administration. Understanding the biological bases for individual differences in cocaine abuse/addiction is of fundamental importance for treating this disease. Individuals vary markedly in their vulnerability to cocaine abuse and addiction. This project will use an animal model to explore how individual differences in normal brain chemistry can contribute to variability in cocaine-induced activation and addiction-like behaviors. Understanding the biological bases for individual differences in cocaine abuse/addiction is of fundamental importance for developing new ways to treat, and ultimately prevent, this devastating brain disease and public health problem.