Charles W. Bradberry - US grants

In both the human clinical situation, and animal models of drug abuse, there are substantial differences between individuals in the amounts of drugs consumed. In an attempt to understand the neurobiology of drug abuse, and relapse following abstention, biochemical correlates of vulnerability to amphetamine self-administration in the rat will be studied. These studies will expand upon recent findings which demonstrate that certain behavioral measures (novelty-and amphetamine-induced locomotion) predict the amount of amphetamine self-administered by animals. Because of the role of the mesoaccumbens dopamine system in supporting both self-administration and amphetamine-induced locomotion, these studies will primarily focus on this system. This proposal is based on the hypothesis that the individual differences between animals which render some more vulnerable to drug self-administration are reflected in measurable biochemical differences. Measurable indices of both pre-and postsynaptic dopaminergic transmission in the nucleus accumbens will be examined. There are four basic aims of this proposal. The first will be to determine to what degree differences in presynaptic dopamine function correlate with differences in novelty-and amphetamine-induced locomotion. Specific experiments will examine amphetamine-induced dopamine release in vivo following local perfusion through a microdialysis probe: 2) amphetamine-induced dopamine release in vitro; 3) whole tissue measures of dopamine and metabolite under basal conditions; 4) dopamine uptake site density. The second specific aim examines mechanisms post-synaptic to dopaminergic actions in the nucleus accumbens for correlation with behavioral predictors: 1) dopamine receptor binding in the accumbens and striatum, 2) dopamine receptor mediated changes in cyclic AMP using accumbens and striatal tissue in vitro, and 3) gamma-aminobutyric acid receptor binding in the ventral pallidum. The third specific aim will determine the correlation between behavioral predictors and the following measures which attempt to determine the "degree of behavioral sensitization" in individual animals: 1) systemic amphetamine-induced dopamine release in vivo, measured by microdialysis in awake rats: 2) morphine-induced locomotion and dopamine release in vivo; 3) stress (novelty) induced increases in extracellular dopamine in the prefrontal cortex and nucleus accumbens. G-protein levels will be determined for correlation with novelty-induced locomotion, novelty-and amphetamine- induced increases in extracellular dopamine, and the effects of pertussis toxin treatment in the ventral tegmental area will be studied. The fourth specific aim will determine if selected parameters (assessed post-mortem) which were shown to correlate with novelty-induced locomotion also correlate with amount of amphetamine self-administered previously in an operant chamber. These studies may substantially enhance our knowledge of the possible biochemical bases of drug abuse and addiction, and therefore increase the prospects for rational therapeutic intervention.

DESCRIPTION (provided by applicant): This renewal will continue our studies on the CNS consequences of chronic cocaine self-administration in rhesus monkeys. The prefrontal cortex, via its interactions with multiple subcortical structures, is intimately involved in reward evaluation and response selection. Human imaging studies of cortical metabolism indicate distinct altered functional states of prefrontal codex at short and long times following cessation of cocaine. There is also evidence of dysfunctionality of prefrontal cortex in cognitive testing and behavioral studies in human cocaine abusers and non-human primates following chronic cocaine. Our studies will determine how altered prefrontal function is manifested via altered subcodical neurotransmission. Specific Aim 1) Characterize the "normal" mode of prefrontal cortex regulation of dopaminergic and serotonergic neurotransmission in the striatum and basolateral amygdala. The impact of electrical stimulation of three key subregions of the prefrontal cortex (anterior cingulate, orbitofrontal, and dorsolateral) on dopamine (DA) and serotonin (5-HT) release in the mesolimbic and sensorimotor striatum and basolateral amygdala will be determined. Specific Aim 2) Determine whether prefrontal cortex regulation of subcodical DA and 5-HT release has been modified in a time-dependent manner - short term cessation of cocaine. Following a minimum of four months chronic cocaine self-administration, the ability of electrical stimulation of subregions of the prefrontal cortex (anterior cingulate, orbitofrontal, and dorsolateral) to stimulate DA and 5-HT release in mesolimbic and sensorimotor striatum and basolateral amygdala wilt be determined. Each study will be conducted 1 day since last cocaine exposure. Specific Aim 3) Determine whether prefrontal cortex regulation of subcortical DA and 5-HT release has been modified in a time-dependent manner - long term cessation of cocaine. Following four months chronic cocaine self-administration, the ability of electrical stimulation of subregions of the prefrontal cortex (anterior cingulate, orbitofrontal, and dorsolateral) to stimulate DA and 5-HT release in mesolimbic and sensorimotor striatum and basolateral amygdala will be determined. Each study will be conducted 3-4 months since last cocaine exposure.

DESCRIPTION (provided by applicant): The aim of this project is to understand inherent differences in serotonin (5-HT) and dopamine (DA) neuronal systems associated with risk for alcoholism. The model to be investigated, the peer-reared rhesus monkey, has significant face validity in terms of clinical correlates of alcoholism: reduced cerobrospinal fluid (CSF) levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), increased consumption of, and tolerance to, ethanol, and increased behavioral impulsivity and aggression. An association of decreased CSF 5-HIAA with impulsivity and high ethanol consumption implicates 5-HT dysfunction, but those functional differences remain unknown. Their impact on ventral striatal DA, also implicated in impulsivity and ethanol reward, are also unknown. The proposed studies will directly examine different aspects of 5-HT and dopaminergic presynaptic function to determine what alterations are associated with reduced CSF 5-HIAA. The specific aims are: 1) Using in-vivo microdialysis, compare the presynaptic activity of the 5-HT innervation of the forebrain in mother reared vs. peer reared rhesus monkeys. Specific assessments of basal extracellular concentration will be determined using the quantitative no-net-flux method in awake, chaired animals. The functionality of the 5-HT transporter will be assessed in the same studies by determining the in-vivo uptake of 5-HT. 2) Conduct brain imaging of the 5-HT transporter in the forebrain and brainstem of mother reared and peer reared rhesus monkeys. We will employ microPET methods with [11C]DASB, a highly selective 5-HT transporter ligand capable of labeling forebrain sites. The imaging outcome measures will be compared with the functional measures of 5-HT transport determined in the same animals in aim 1.3) Compare the responsiveness of ventral striatal DA and cortical 5-HT to acute ethanol using microdialysis in awake mother reared and peer reared animals. 4) Compare post-mortem whole tissue levels 5-HT, 5-HIAA, the 5-HT transporter, DA (and its metabolites), and monoamine oxidase activity in cingulate cortex and ventral striatum of mother reared and peer reared animals. The values will be compared across individuals with the imaging and microdialysis measures from aims 1, 2 and 3. Unused brain tissue will be banked for future potential uses

The overall goal of this project is to provide a bridge between the animal studies proposed in the Center on D 1 receptor modification of cortical function with the clinical studies proposed in project 9. Our working hypothesis is that D1 receptor modulation causes state-dependent secondary alterations in the function of other key neurotransmitters such as glutamate, GABA, DA, and serotonin in the prefrontal cortex. Hence, we propose to examine the effect of focal or systemic D1 receptor manipulation, administered in a parallel manner to both the electrophysiological and clinical studies proposed in other projects of this Center, on the release of cortical DA, serotonin, GABA, and glutamate while the animal is under various states that include (1) rest, (2) cognitive activation caused by engagement in working memory task, and (3) cognitive impairment caused by chronic neuroleptic treatment. The specific aims are: 1) Determine the effect of intracortical application of a D1 agonist and antagonist on extracellular efflux of glutamate, GABA, DA, and serotonin in awake, chaired animals at rest or engaged in a working memory task, or a control visually guided task. Based on in vitro and in-vivo analysis of D1 actions, we hypothesize that the D1 agonists will reduce glutamate, GABA, DA and serotonin release. 2) Determine the effect of acute and subchronic systemic treatment with DAS-431 (the D1 agonist to be employed clinically in project 7) on extracellular efflux of glutamate, GABA, DA, and serotonin in chaired animals at rest, engaged in a working memory task, or a control visually guided task. 3) Determine the acute and subchronic effect of DAS-431 on extracellular efflux of glutamate, GABA, DA, and serotonin in animals that have received chronic neuroleptic treatment. Studies will be performed at rest, engaged in a working memory task, or a control visually guided task.

DESCRIPTION (provided by applicant): Cognitive dysfunction and impaired inhibitory control are hallmarks of addiction. The prefrontal and temporal cortices are essential to effective cognitive performance, and drug abuse is associated with significant structural deficits therein. A crucial unresolved question in clinical studies is whether structural and functional differences in cortex predate, or are consequent to drug use. This application proposes longitudinal structural MR imaging in a monkey model of addiction-related cognitive and inhibitory control deficits to address the role of drug use per se in structural and functional cortical differences seen in cocaine addiction. It also will examine single unit activity to determine what cellular changes could mediate an association of altered structure and function. Animal models are key to addressing questions about the etiology and cellular basis of addiction-related cognitive dysfunction. This is especially so for primate models that share structural and cognitive similarities to humans at the cortical level and that can exploit common methodologies such as brain imaging techniques used clinically. This application will employ a clinically relevant rhesus monkey self- administration model that shows impaired performance virtually identical to that seen clinically on the Stop Task, which is used to measure impaired inhibitory control, and which is supported by a well- defined circuitry identified in clinical and pre-clinical studies. Deficits in stimulus discrimination consistent with clinical reports have also been established. We will employ the monkey model to address these fundamental questions: 1) Do structural alterations in prefrontal and temporal cortex observed in cocaine users result from drug exposure per se, rather than a preexisting condition? 2) Within individuals, does the degree of cognitive impairment correlate with extent of structural change? 3) What are cellular correlates of cognitive dysfunction associated with cocaine use? The integration of a longitudinal application of clinically employed structural and cognitive assessments, along with single unit studies, will help establish the relationship between cocaine use, altered structure, and cellular mechanisms associated with cognitive dysfunction observed clinically. Drug addiction causes extensive human suffering and financial loss to society. Understanding the source and mechanisms of cognitive dysfunction that predicts treatment outcome may help develop therapeutic approaches that lessen harm from addiction.

Abstract: This application seeks funding to address the opportunity at the University of Pittsburgh for a coherent training program on the neurobiology of substance use and abuse. The program will support three post- doctoral trainees and three pre-doctoral trainees at the dissertation stage. It is structured around five core areas of training faculty expertise: 1) Systems neurobiology of motivated behavior and cognition 2) Adolescent developmental biology and substance use vulnerability 3) Molecular modulation of neurotransmitter release and reuptake 4) Nicotine reinforcement and relapse 5) Stress responsive brain circuits. A didactic course will address these core areas of focus. There will also be a formal seminar series drawing prominent scientists whose work is at the cutting edge of research into these areas of focus. Extensive training in grant writing is formally incorporated through a mentored and reviewed grant writing course requirement. Presentation and analysis skills will be developed through an in-house seminar series in which works in progress will be presented by trainees or other individuals within the University of Pittsburgh substance use research community. The training program will enhance critical skills necessary for success as independent investigators, and the sum of its components will create multiple nodes of interaction between researchers, thereby increasing the breadth and quality of training of the supported individuals.

DESCRIPTION (provided by applicant): This is a new RO1 entitled Neurodegeneration in aged SIV-infected primates that is a direct extension of our previously investigations in the pathogenesis of SIV encephalitis and neurodegeneration in aged non-human primates. Use of combined active antiretrovirals (CART) in developed countries has led to a near disappearance of severe encephalitis. Unfortunately chronic HIV infection continues to exact a toll on the nervous system with increased prevalence of a spectrum of neurocognitive and motor dysfunctions termed HIV-associated neurocognitive disorders (HAND). CART has also permitted people to survive longer with HIV infection and the CDC projects that by 2015 over half of HIV infected individuals in the US will be over the age of 50. Coupled with the aging process, the extended exposure to both HIV and antiretroviral drugs appears to increase their risk of neurologic and neuropsychiatric complications. In this application we propose to use a well-established non-human primate (NHP) model of chronic lentiviral infection, SIV infection of Macaca mulatta (Rhesus Macaque RM), to model HAND in aged macaques (>20 years old), map the neurological signs to behavioral abnormalities and begin to elucidate the neurological and immunological pathogenesis of this debilitating disease. While no animal disease model is perfect, numerous similarities between simian and human nervous systems, between SIV and HIV infection and the capacity to manipulate and monitor central nervous system (CNS) damage, make the macaque model optimal for these studies. Using 5 groups of SIV infected and control RMs, we will assess the presence of neurocognitive abnormalities and the role of viral suppression in exacerbating age related neurodegeneration. Findings from this grant will have immediate implications on the treatment of aged HIV infected humans. Building upon our previous decade of experience with SIV infection of macaques as a model of chronic HIV infection and our recent studies of neuroinflammation in aged NHPs (Kofler et al 2011 (41) see appendix), we will test our overarching hypothesis that: chronic systemic CART with or without chronic lentiviral infection exacerbates age related damage to the nervous system principally through CNS stress and associated innate immune activation. We will assess whether aggressive suppression of systemic lentiviral infection with CART exacerbates age related cognitive abnormalities and beta amyloid related neuropathology (e.g. plaques, tangles, abnormal tau phosphorylation or beta amyloid oligomeres). Alternatively, HAND may persist even in the presence of viral suppression or as a result of CART alone. In our second specific aim we will use state of the art quantitative neuropathological analysis to elucidate the pathological substrate of HAND. Knowledge of the pathogenesis of neurological dysfunction in the simian model will help define pathways for intervention to mitigate the human disease. PUBLIC HEALTH RELEVANCE: This application will develop a monkey model to help elucidate mechanisms of HIV-associated neuropathogenesis in the context of aging, chronic infection with HIV, and long-term exposure to Highly Active Antiretroviral Therapy. Defining the disease mechanisms linking brain aging, lentiviral infection and anti- retroviral therapy is essential to design treatments to avoid adverse neurological consequences of HIV infection in the middle age and elderly. !