Glen R. Hanson - US grants

The abuse of the amphetamine analog, 3,4-methylenedioxymethamphetamine (MDMA), continues as a significant public health problem. Treatment of rats with MDMA destroys brain serotonergic nerve terminals which is accompanied by a long-lasting decrease in tryptophan hydroxylase (TPH) activity. The mechanism by which this neuronal damage occurs remains unknown. Interestingly, MDMA causes a biphasic response in the central serotonergic system. The first phase consists of a rapid decrease in the cerebral TPH activity which is detected within 15 min of drug administration. This early phase (phase l) is reversed by incubating the enzyme in vitro under reducing conditions. In the second phase, a decrease in TPH activity (phase 2) is observed days after MDMA administration and last for extended periods of time; in contrast to phase l, phase 2 cannot be reversed by reducing conditions. The long-lasting change in TPH activity is associated with MDMA-induced neurotoxicity. Evidence presented in this proposal suggests that TPH protein undergoes an initial reversible transformation during phase 1, with other possible transformations during an intermediate transitional stage occurring prior to TPH protein destruction due to neuronal damage (phase 2). The phase l decrease in TPH activity is caused by oxidative conditions which are proposed to predispose the serotonergic neuron to permanent neuronal (damage. Since TPH is sensitive to MDMA-induced oxidative alterations present in the serotonergic system, we propose to use this enzyme as a probe to monitor and characterize this phenomenon. The characterization of these oxidative conditions will be achieved by identifying the different stages of TPH protein transformation by correlating the MDMA-induced decrease in TPH activity with its protein concentration and with recovery of enzyme activity induced by reducing conditions. The MDMA-generated oxidative conditions will be further characterized by studying the effects of different free radical scavengers on the three stages of TPH transformation. The role of glutathione as a protective system in the serotonergic system will also be investigated. Finally, the reactive substances generated by MDMA will be investigated by studying the alterations of the TPH protein isolated during the different stages by immunoprecipitation and electrophoresis. This study should assist in identifying the biochemical alterations induced by MDMA and similar amphetamine analogs and their impact on brain proteins.

DESCRIPTION: (Applicant's Abstract) Problems associated with methamphetamine (METH) abuse have increased dramatically in the U.S. due to its escalating illicit use. To elucidate the responses to this drug and possibly identify novel therapeutic strategies for dealing with these problems, the relationship between extrapyramidal neuropeptide systems and the effects of this potent stimulant has been studied. It was observed that a low dose of METH (0.5 mg/kg) selectively influenced neurotensin (NT) systems in the dorsolateral caudate by activating dopamine D-2 receptors. In contrast, high doses of METH (10-15 mg/kg) selectively altered NT systems associated with the medial/ventral caudate by activating dopamine D-1 receptors. Because D-2 and D-1 dopamine receptors are selectively expressed in the striatal-pallidal and striatal-nigral neurons, respectively, these findings led to the hypothesis that low and high doses of METH preferentially influence the indirect (striatal-pallidal) and direct (striatal-nigral) efferent pathways to the basal ganglia output nuclei, respectively. This hypothesis will be tested by achieving the following Specific Aims: A. Determine if low doses of METH preferentially alter the activity of the indirect striatal efferent pathway to the globus pallidus. The effects of low doses of METH on associated transmitter systems (i.e., NT, met-enkephalin [M-enk] and GABA) will be monitored by measuring drug-induced changes in neurotransmitter (a) tissue content (NT and M-enk), (b) precursor mRNA levels (NT and M-enk), and (c) in vivo release (NT, M-enk and GABA). The role of the D-2 receptor in these effects will be elucidated. B. Determine if high doses of METH preferentially alter the direct striatal efferent projection to the substantia nigra. The effects of high doses of METH on associated transmitter systems (i.e., NT, substance P [SP] and GABA) will be monitored by measuring drug-induced changes in neurotransmitter (a) tissue content (NT and SP), (b) precursor mRNA levels (NT and SP), and (c) in vivo release (NT, SP and GABA). The role of the D-1 receptor in these effects will be elucidated. In addition, the role of striatal cholinergic interneurons in the effects identified from Specific Aims A and B will be studied. These studies may lead to a better appreciation of the neurobiology of these systems and improved therapies for problems associated with METH abuse as well as extrapyramidal/limbic dysfunctions.

DESCRIPTION: (Applicant's Abstract) This is a Senior Scientist Award proposal for Dr. Glen R. Hanson. Dr. Hanson is a tenured professor in the Department of Pharmacology and Toxicology at the University of Utah. He has been active in drug abuse research for over 15 years and has made important contributions to elucidating: (a) the role of dopamine systems in neurotoxicity caused by amphetamine analogs; (b) the role of reactive oxygen species in mediating monoaminergic changes caused by amphetamine analogs; (c) the influence of the stimulants of abuse on neuropeptide systems; (d) the role of dopamine receptor subtypes in the regulation of extrapyrarnidal and limbic neuropeptide systems. Dr. Hanson currently devotes 40-50% of his time to research and student mentoring while 50-60% of his effort is occupied by teaching, departmental and college responsibilities. This Senior Scientist Award will allow Dr. Hanson to increase his research effort to approximately 80%. During the time of support by this award, he will direct research which tests the following principal hypotheses: (a) the generation of dopamine-related reactive oxygen species is a major contributor to the neurochemical deficits occurring in the striatal monoaminergic systems after high doses of methamphetamine (METH); (b) methcathinone, a relatively new designer amphetamine, exerts profound short-and long-term effects on extrapyramidal and limbic monoaminergic systems and has significant neurotoxic potential; (c) METH acutely and selectively alters the nature and function of the dopamine transporter in a reversible manner; (d) low and high doses of METH preferentially influence the indirect (striatal-pallidal) and direct (striatal-nigral) efferent pathways to the basal ganglia output nuclei, respectively; (e) changes in neuropeptide systems occur in humans exposed to the potent stimulants of abuse much like that observed in rats after treatment with these same drugs. The increase in research time resulting from a Senior Scientist Award will allow Dr. Hanson to develop expertise in technologies new to his laboratory required to test these hypotheses, such as: (a) free radical analysis; (b) transporter function and ligand assays; (c) in situ hybridization for neuropeptide precursor mRNA. This award will help Dr. Hanson to mentor more effectively inexperienced researchers at the undergraduate, graduate, postdoctoral and young faculty levels as these developing scientists mature and establish themselves in neuroscience research in general, and drug abuse research in particular. In addition, as a mentor Dr. Hanson will be especially supportive of the involvement of underrepresented minorities in drug abuse research.

[unreadable] DESCRIPTION (provided by applicant): This is a competitive renewal application for Dr. Glen Hanson's Senior Scientist Award. Dr. Hanson is a tenured professor in the Department of Pharmacol. & Tox. at the University of Utah with more than 20 years experience in drug abuse research resulting in important contributions to our understanding of the short- and long-term effects of psychostimulants on brain monoaminergic and neuropeptide systems. Dr. Hanson's K05 award (beginning in 1998) has relieved him from heavy teaching and committee responsibilities, allowing Dr. Hanson to devote > 75% of his professional time to substance abuse/addiction research-related endeavors. Activities supported by his K05 award resulted in: (i) ~57 peer-reviewed scientific publications; (ii) >90 abstracts presented at scientific meetings; (iii) an appointment as the director of NIDA's Division of Neuro-science and Behavioral Research (2000-2001); (iv) becoming the acting Director of the National Institute on Drug Abuse (2001-2003); (v) an appointment as the director of the Utah Addiction Center (2003-present); and (vi) becoming a senior advisor to the director of NIDA (2003-present). Renewal of Dr. Hanson's K05 will permit him to direct research to test the following principal hypotheses: (i) the functional output of the striato-nigral pathway is increased by administration of low doses of methamphtamine and unaffected by administration of high doses of this drug; (ii) expression of a 2nd , but not the 1st , component of an acute response of the vesicular monoamine transporter-2 is linked to the occurrence of phenylethylamine-induced monamine neurotoxicity; and (iii) there are related mechanisms fundamental to the refractoriness of the nigrostriatal dopamine pathway to methamphetamine-induced neurotoxicity in: adolescent; previously lesioned; and tolerant, rats. Continuation of his K05 will allow Dr. Hanson to develop expertise in technologies that will substantially further his research efforts, such as: (i) subcellular fractionation; (ii) Western blotting and immunoprecipitation protein characterization; (iii) rotating disk electrode voltammetry; and (iv) self-administration contingency paradigms. In addition to research projects, the K05 support will allow Dr. Hanson to direct efforts to create interdisciplinary research teams to conduct drug abuse and translational research. Finally, this award will permit Dr. Hanson to continue mentoring inexperienced researchers at the under-graduate, graduate, postdoctoral and young faculty levels, to assist them in their scientific development. [unreadable] [unreadable] [unreadable]

Because abuse and associated problems of the phenylethylamine drugs, such as methamphetamine (METH), continue to be a major concern in the United States and throughout the world, it is critical to under- stand the short- and long-term consequences resulting from abusing these psychostimulants. Of particular relevance to this project is the elucidation of the role of monoamine transporters to the acute andchronic effects of phenylethylamines. We recently demonstrated that METH treatments rapidly and profoundly reduce the activity of vesicular monoamine transporters-2 (VMAT-2) in striatal purified vesicles. This VMAT- 2 Acute Transporter Response (VATR) has at least two distinct components: one (1st component) that occurs after a single METH administration, and the other (2nd component) apparently only after multiple METH adminstrations that lead to neurotoxicity. Based on these observations, we hypothesize that "Expression of a 2nd, but not the 1st, component of the VATR is linked to occurrence of phenylethylamine- induced monoamine neurotoxicity." This hypothesis will be tested by achieving the following Specific Aims: A. Determine and compare the features of the components of the METH-induced striatal VATR. We will study and contrast the full complement of basic dose, temporal, kinetic, anatomical, and mechanistic features of the VATR after a single (nontoxic) and multiple (neurotoxic) METH administrations. B. Elucidate the funtional significance of the VATR components. This will be accomplished by determining if the VATR components interfere with vesicular monoamine sequestration and releasing functions. C. Determine the effects on VATR expression of diverse phenylethylamines (i.e., amphetamine, MDMA, fenfluramine, and ephedrine), with monoamine neurotoxcity profiles distinct from METH. This will identify how the VATR components link with the various effects on monoamine systems and different expressions of monoamine toxicity associated with these distinct phenylethylamine analogs.

DESCRIPTION (provided by applicant): The continued abuse of methamphetamine (METH) in this country is particularly troubling because of the severe persistent psychiatric symptoms and disturbing social impact of this potent psychostimulant. In order to better deal with the consequences of METH addiction, it is important to elucidate the mechanisms associated with its short- and long-term effects. Two important issues we and others have researched that are particularly germane to the present proposal are: first, the property of METH to cause persistent deficits in striatal dopamine systems (neurotoxicity) under some, but not all, conditions: and second, the related observation of a unique response to METH treatment by adolescent versus adult animals. The overall objective of this revised proposal is to elucidate the neurotoxic responses of subcortical monoamine systems to METH, as it relates to the differential vulnerability of striatal dopamine (DA) under varying refractory conditions. Thus, studies in this proposal are based on our and others' observations that factors linked to the METH-induced persistent striatal DA deficits fall into two stages based on their temporal relationship to drug exposure (i.e., Stage 1=0-8 h and Stage 2=24-72 h). We will achieve the overall objective by testing the prevailing hypothesis that events occurring during Stage 1 followed by those of Stage 2 are necessary for the expression of METH-induced toxicity in striatal DA systems and the models of resistance to METH toxicity to be studied in this Program Project interfere with the requisite events in distinct ways. This hypothesis will be tested by examining alterations in the expressions of METH-induced Stages 1 and 2, and underlying mechanisms, in the following models of METH resistance: (i) adolescent rats (project #1- Developmental Refractoriness, project director is Dr. Annette Fleckenstein); (ii) adult rats pretreated with a neurotoxic-METH treatment (project #2-Lesion-induced Refractoriness in Adults, project director is Dr. Kristen Keefe); and (iii) rats previously exposed to a temporary tolerance caused by incrementally increasing METH doses (project #3-tolerance-related Reversible Refractoriness, project director is Dr. Diana Wilkins). It is anticipated that elucidation of mechanisms for these 3 refractory models will inform issues such as: (i) adolescent drug abuse vulnerability; (ii) impact of METH use during adolescent development; and (iii) the persistent impact of METH-related neurotoxicity during adulthood. PROGRAM PROJECT CHARACTERISTICS

DESCRIPTION (provided by applicant): Methamphetamine (METH) alters monoaminergic (e.g. dopaminergic; DA) systems leading to a refractory dependence and psychotic, violent and criminal behaviors. Because no FDA-approved medications are available to treat METH dependence, the National Institute on Drug Abuse (NIDA) has encouraged research of CNS systems that contribute to METH dependence to identify novel therapeutic strategies. Thus, others and we study how neurotensin (NT) influences the function of DA basal ganglia and limbic pathways and how it contributes to the effects of METH. NT is a neuropeptide associated with both the indirect (D2-regulated) and direct (D1-regulated) feedback pathways to the nigrostriatal DA projection, with similar, but less well delineated, feedback arrangements in limbic structures. Overall stimulation of these NT systems reduces DA-mediated behaviors and counteracts overactive DA pathways. Consequently, these NT pathways have been classified as natural neuroleptic systems and NT agonists have been suggested by NIDA as possible medications for treating drug abuse. It has been reported that not only does NT mitigate DA responses, but its pathways are reciprocally regulated by DA with stimulation of D2 and D1 receptors causing opposing decreases and increases of NT tissue levels, respectively, in both basal ganglia and limbic brain regions. Relevant to the present proposal are findings that similar NT responses to non-contingent low and high doses of METH are principally mediated by these same D2 and D1 receptors, respectively. In order to determine the clinical relevance of these findings, we used METH self-administration (SA) models based on contingent lever pressing to obtain METH infusion and observed that: (i) the NT agonist, PD149163 blocks METH SA while not substituting for METH nor being self-administered per se; (ii) PD149163 blocks lever pressing during maintenance (i.e., operant responses associated with stable METH SA), beginning of extinction (i.e., elimination of lever pressing when no longer linked to METH infusion) and reinstatement (the lever- pressing response of rats to a METH trigger given after extinction); (iii) endogenous NT systems linked to D2 and D1 mechanisms, differentially contribute to behaviors associated with extinction and maintenance, respectively. Based on these findings, we will examine behavioral and basal ganglia and limbic NT/DA responses to test the hypothesis that endogenous NT systems have differential roles in extinction, maintenance and reinstatement of METH SA, by achieving the following: Specific Aim A: Determine the role of NT systems and related D2 receptors on extinction of METH- seeking behavior (i.e., reduced lever pressing). Specific Aim B:Determine the role of NT systems and related D1 receptors on METH SA maintenance. Specific Aim C: Determine the effect of METH SA reinstatement on NT systems. PUBLIC HEALTH RELEVANCE: Methamphetamine (METH) abuse inflicts severe personal and societal harm. Our extensive studies have suggested that basal ganglia and limbic neurotensin (NT) systems are sensitive to dopamine (DA) D1 and D2 regulation and in turn through inhibitory feedback mechanism help to influence the response in rats to non- contingent METH treatment. To determine the clinical relevance of these findings to METH abuse, we are studying the impact of METH self-administration (contingent on lever pressing) on the NT pathways associated with the dorsal striatum and nucleus accumbens. We have determined that NT systems respond in contrasting ways to maintenance, extinction, and reinstatement in our self-administration METH model and that endogenous NT systems specifically contribute to the elimination of lever pressing during extinction as well as a lack of lever pressing in a minoriy of non-responding rats during maintenance. Because of these findings we propose to test the specific hypothesis that endogenous NT systems have differential roles in the extinction, maintenance, and reinstatement of METH self-administration.