Rosemarie M. Booze - US grants

Cocaine is undergoing an epidemic of abuse in the US; however, little is known concerning the neural mechanisms which lead to the abuse of stimulants. Using a neuroanatomical approach, these studies will test the hypothesis that cholinergic interneurons represent an integral pathway in nucleus accumbens. This pathway is likely to be critical for the expression of the reinforcing properties of cocaine and other stimulants. Progress along these lines will contribute important knowledge to the broader issue of the neurobiological substrates underlying drug abuse and addiction. This type of information will provide the basis for targeting multiple pharmacological sites and devising rational therapeutic strategies. The specific aims of the proposal are: 1) To test the ability of cocaine to modulate dopamine receptors in the nucleus accumbens and striatum. Quantitative receptor autoradiography will be used to examine the dopamine D-1 and D-2 receptor subtypes in the nucleus accumbens after acute and repeated cocaine administration. These findings will confirm and extend prior reports that the nucleus accumbens possesses a unique neural response to cocaine. 2) To examine whether lesions of the allocortical afferent pathways to the nucleus accumbens affect the D-2 dopamine receptor response to cocaine. Afferent pathways to the nucleus accumbens from the hippocampus and amygdala will be lesioned in order to determine whether the D-2 binding sites are localized to intrinsic neural populations. It is predicted that lesions of afferent pathways will not alter the D-2 receptor response to cocaine. 3) To demonstrate that interneurons of nucleus accumbens are the site of cocaine-induced changes in D-2 receptor loss. It is predicted that the D-2 receptors which are localized to intrinsic neurons of the nucleus accumbens will be lost, thereby abolishing the response of D-2 receptors to cocaine. 4) To determine whether the effects of cocaine of D-2 receptors are linked to cholinergic interneurons in the nucleus accumbens. In situ hybridization for D-2 receptor mRNA and immunocytochemical staining of cholinergic and GABAergic interneurons will be combined to directly identify the cellular localization of D-2 receptors. It is predicted that D-2 receptors will be localized to cholinergic interneurons in the nucleus accumbens. These studies will examine the regulation of the D-2 receptor by cocaine at the molecular (mRNA) level.

The long-term goal of this project is to determine the mechanism of selective neuronal death in aging and AD. This project tests the hypothesis that glucocorticoids interact with calcium regulatory proteins, producing neural dysfunction and cell death. This hypothesis arises from studies in aged animals demonstrating that prolonged exposure to glucocorticoids contributes to age-related neuronal cell death as well as from studies linking altered calcium homeostasis to age-related neural dysfunction. A more general version of this hypothesis is to be examined: an increase in the ratio of proteins that favor an increase in intracellular [Ca] (e.g., GCRll and L-type calcium channels) relative to those proteins that reduce, buffer or extrude calcium (e.g., Ca, Mg-ATPase, protein kinase C and calbindin-D28k) occurs in brain cells that are most at risk for degeneration in aging and AD. This project will specifically address 1) the neuroanatomical co-localization and quantitative ratio of GCRll relative to calcium regulatory proteins in the aging rat hippocampus, 2) quantitatively analyze age-related changes in mRNA expression for GCRll and calcium regulatory proteins in rat hippocampus, 3) test the acute and long-term regulation of GCRll and calcium regulatory proteins as a function of aging, and 4) determine potential interactions between intracellular calcium, calcium regulatory proteins and GCRll in the dysfunction of AD. Progress along these lines of research will test a novel hypothesis in AD - the glucocorticoid/calcium dyshomeostasis hypothesis of cell death, in addition to testing the glucocorticoid system and the calcium regulatory system independently. Even if these two systems are not linked, the proposed studies will yield the first systematic and comprehensive examination of two presently viable hypotheses (GCs and Ca regulation) of age-related neuronal loss in relation to specific pathological alterations in aging and the AD brain.

DESCRIPTION: (Applicant's Abstract) Gender differences in response to psychostimulants have been reported both in animals and humans; however, the biological mechanisms which underlie these gender differences to psychostimulants remain for the most part, unexplained. The common observation is that females are more sensitive to psychostimulants, such as cocaine. Our hypothesis is: Activational as well as organizational effects of gonadal hormones on dopaminergic systems provide the underlying basis for the gender differences in behavioral sensitization produced by repeated IV cocaine administration. First, we will determine whether pharmacokinetic differences between the sexes result in higher levels of cocaine in the female brain. We have successfully developed a technically simple, economical and practical non-tethered technique for repeatedly administering cocaine IV to freely moving, group-housed, rats. Detailed pharmacokinetic analysis has demonstrated rapidly peaking cocaine levels following IV dosing in rats, which is similar to that observed in humans, as opposed to SC, PO, or IP dosing. Using this clinically relevant IV rodent dosing model, we will determine whether pharmacokinetic factors contribute to the increased sensitivity of female animals to the effects of cocaine. Second, we will determine whether gonadal hormones regulate the expression of gender differences in response to cocaine in adulthood. We will test the ability of gonadal hormones to modulate dopamine receptor responsiveness to chronic cocaine administration. Third, we will determine whether the brain organizational effect of the perinatal hormonal milieu mediates the gender differences in cocaine responsiveness. We have pharmacologically characterized a recently discovered unique dopamine receptor subtype (D3) which is localized to the striatum/nucleus accumbens region of the brain. We hypothesize that alterations in dopaminergic systems, in particular the D3 receptor system, underlie the gender differences produced by repeated IV cocaine administration. Our long-term goal is to determine the role of the dopamine neurochemical system (emphasizing the dopamine D3 receptors) in gender differences following repeated IV cocaine administration. The ultimate goal of this research is to develop pharmacological interventions to assist in correcting the behavioral problems associated with chronic cocaine abuse in humans, and specifically to provide potential insight into effective treatment strategies for women drug abusers.

Women now constitute one of the fastest growing populations becoming infected with HIV. Drug use plays a major role in the spread of this disease in women: 46 percent of women's AIDS cases are directly attributable to injection drug use, vs. only 22 percent of cases in men. In addition, gender differences in response to psychostimulants (i.e., females are more sensitive to cocaine and amphetamines) have been reported both in animals and humans; however, the biological mechanisms which underlie these gender differences to HIV infection and psychostimulants remain for the most part, unexplained and undressed scientifically. The proposed program poses the major question: are biological gender differences n responsiveness to (repeated) cocaine predisposing females ot HIV-induced neurotoxicity? Our hypothesis is: Estrogens will act as protective agents fot he combined effects of repeated IV cocaine administration and gp120/tat neurotoxicity. First, we will determine whether steroid hormones are neuroprotective against the combined effects of gp120/tat and cocaine in cultured human fetal neurons. Second, we will determine whether estrogen regulate the expression of gender differences in response to HIV neurotoxicity in adulthood. Specifically, we will test the ability of estrogen to modulate gp120 and tat neurotoxicity in female animals chronically exposed to cocaine. We have successfully developed an innovative, technically simple, economical and practical on-tethered technique for repeatedly administering cocaine IV to freely moving group-housed, rats. Detailed pharmacokinetic analysis has demonstrated rapidly peaking cocaine levels following IV dosing in rats, which is similar to that observed in humans. Using this clinically relevant IV rodent dosing model, we will determine whether pharmacokinetic factors contribute to the increased sensitivity of female animals to the effects of nicotine. Third, using this clinically relevant IV cocaine rodent-dosing model, we will determine whether gp120 and tat produce dopaminergic neurotoxicity and receptor alterations in female animals following chronic cocaine. Our rodent model of IV cocaine, in combination with HIV protein neurotoxicity, is innovative and will be translational to the important woman's health issues of drug abuse and AIDS. The goal of this research is to provide potential insight into effective biologically-based gender-specific treatment strategies for HIV and cocaine addiction.

[unreadable] DESCRIPTION (provided by applicant): Gender differences in response to psychostimulants have been reported both in animals and humans; however, the biological mechanisms which underlie these gender differences to psychostimulants remain for the most part, unexplained. The common observation is that females are more sensitive to psychostimulants, such as nicotine. Our hypothesis is: Gonadal hormones in adulthood and development act on dopaminergic systems, providing the underlying basis for the gender differences in behavioral sensitization produced by repeated IV nicotine administration. First, we will determine whether pharmacokinetic differences between the sexes result in higher levels of nicotine in the female brain. We have successfully developed a technically simple, economical and practical non-tethered technique for repeatedly administering IV nicotine to freely moving, group-housed rats. Detailed pharmacokinetic analysis has demonstrated rapidly peaking nicotine levels following IV dosing in rats, which is similar to that observed in humans, as opposed to SC or PO dosing. Using this clinically relevant IV rodent dosing model, we will determine whether pharmacokinetic factors contribute to the increased sensitivity of female animals to the effects of nicotine. Second, we will determine whether gonadal hormones regulate the expression of gender differences in response to nicotine in adulthood. We will test the ability of gonadal hormones to modulate dopamine receptor responsiveness to chronic nicotine administration. Third, we will determine whether the brain organizational (neurodevelopmental) effect of the perinatal hormonal milieu mediates the gender differences in nicotine responsiveness. We have pharmacologically characterized a recently discovered unique dopamine receptor subtype (D3) which is localized to the striatum/nucleus accumbens region of the brain. We hypothesize that alterations in dopaminergic systems underlie the gender differences produced by repeated IV nicotine administration. Our long-term goal is to determine the role of the dopamine neurochemical system in gender differences following repeated IV nicotine administration. The ultimate goal of this research is to develop pharmacological interventions to assist in correcting the behavioral problems associated with chronic tobacco use in humans, and specifically to provide potential insight into effective gender-specific treatment strategies for smoking cessation.

DESCRIPTION (provided by applicant): Although combination antiretroviral therapy (CART) has been highly successful in reducing mother-to-child- transmission (MTCT) in the U.S., globally, MTCT is presently responsible for >1000 new HIV-1 infections each day or more than one new pediatric infection every two minutes. The dopamine (DA) system is a clinically relevant target as evidenced by recent imaging, neurocognitive, and post-mortem examinations of the HIV-1 infected patients. Using a prospective longitudinal design, in this competing renewal we will explore the hypothesis that the development and progression of neurocognitive dysfunction associated with HIV-1, is consequent of, and attributable to, pathology of the DA system, a system highly sensitive to inflammatory processes. Longitudinal studies, while challenging in non-rodent species, are critical for systematically addressing pediatric HIV-1/AIDS, and are also fundamental to our understanding of chronic HIV-1 associated neurological disorders (HAND). The specific aims are: 1) To determine the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. Developmental milestones and an array of tasks directed at the behaviors expressing the prominent components of neurocognitive dysfunction that have been clinically identified will be assessed during the rodent preweaning period, adolescence, adulthood and middle age. 2) To determine the long-term alterations in the major DA receptor subtypes and DA transporter as a candidate neurochemical mechanism for the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV- 1 protein exposure. Quantitative autoradiography will be used to assess the expression of DA receptors (D1, D2, and D3) and DA transporters (DAT) in the nigrostriatal and mesocorticolimbic pathways of the DA system during adolescence, adulthood and middle age. 3) To determine the integrity of DAT function in the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. DAT function will be studied in a transgenic rat model of chronic HIV-1 protein exposure using in vivo NNF/microdialysis. 4) To determine whether the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure may be treated with currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction. These experiments will establish the functional role of the neurobiological changes in the DA receptors and DAT as a neurochemical mechanism contributing to, if not mediating, the neurocognitive impairments consequent to chronic expression of the HIV-1 transgene. The program goal is to advance the field with a translational model of the core components of cognition relevant to pediatric HIV-1/AIDS as well as to HAND, and more importantly, to identify (normal rats) and validate (the better characterized HIV-1 transgenic rat) novel neurotherapeutics to tune the cognition domains afflicted by HIV-1.

DESCRIPTION (provided by applicant): Although combination antiretroviral therapy (CART) has been highly successful in reducing mother-to-child- transmission (MTCT) in the U.S., globally, MTCT is presently responsible for >1000 new HIV-1 infections each day or more than one new pediatric infection every two minutes. The dopamine (DA) system is a clinically relevant target as evidenced by recent imaging, neurocognitive, and post-mortem examinations of the HIV-1 infected patients. Using a prospective longitudinal design, in this competing renewal we will explore the hypothesis that the development and progression of neurocognitive dysfunction associated with HIV-1, is consequent of, and attributable to, pathology of the DA system, a system highly sensitive to inflammatory processes. Longitudinal studies, while challenging in non-rodent species, are critical for systematically addressing pediatric HIV-1/AIDS, and are also fundamental to our understanding of chronic HIV-1 associated neurological disorders (HAND). The specific aims are: 1) To determine the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. Developmental milestones and an array of tasks directed at the behaviors expressing the prominent components of neurocognitive dysfunction that have been clinically identified will be assessed during the rodent preweaning period, adolescence, adulthood and middle age. 2) To determine the long-term alterations in the major DA receptor subtypes and DA transporter as a candidate neurochemical mechanism for the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV- 1 protein exposure. Quantitative autoradiography will be used to assess the expression of DA receptors (D1, D2, and D3) and DA transporters (DAT) in the nigrostriatal and mesocorticolimbic pathways of the DA system during adolescence, adulthood and middle age. 3) To determine the integrity of DAT function in the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. DAT function will be studied in a transgenic rat model of chronic HIV-1 protein exposure using in vivo NNF/microdialysis. 4) To determine whether the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure may be treated with currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction. These experiments will establish the functional role of the neurobiological changes in the DA receptors and DAT as a neurochemical mechanism contributing to, if not mediating, the neurocognitive impairments consequent to chronic expression of the HIV-1 transgene. The program goal is to advance the field with a translational model of the core components of cognition relevant to pediatric HIV-1/AIDS as well as to HAND, and more importantly, to identify (normal rats) and validate (the better characterized HIV-1 transgenic rat) novel neurotherapeutics to tune the cognition domains afflicted by HIV-1.

? DESCRIPTION (provided by applicant): Despite the widely acknowledged success of combination antiretroviral therapy (cART) in the incidence of HIV- 1 associated dementia (HAD), HIV-1-associated neurocognitive disorders (HAND) continue to afflict up to 50% of patients on cART. Neurological complications of HIV infection are the biggest challenge facing HIV researchers, and there is a critical need to develop treatment approaches for HAND. Using the HIV-1 transgenic (Tg) rat model in a prospective longitudinal design, we will explore the hypothesis that the progression of neurocognitive dysfunctions associated with HIV-1 are consequent to central nervous system (CNS) synaptodendritic pathology, neuroinflammation and oxidative stress, and that the progressive neurocognitive losses will be slowed or halted with a restorative therapeutic approach administered early in the course of transgene expression. The specific aims are: 1) To establish the progression of neurocognitive decline in the HIV-1 Tg rat on measures of attention and executive function; as distinct from potential changes in sensory, activity, and motivational confounds. The progression of neurocognitive dysfunction will be assessed with periodic tests from pre-adolescence through advanced age, given the exponential growth of 65+ year old HIV+ patients. Both males and females will be examined. 2) To establish the progression of CNS synaptodendritic pathology, neuroinflammation and oxidative stress, in the HIV-1 Tg rat. Investigations will focus on quantifying dendritic branching and spine parameter alterations with respect to the prefrontal cortex and medium spiny neurons of the nucleus accumbens. 3) We will protect (and restore) neurocognitive function and synaptodendritic complexity with S-equol, a metabolite produced via the gut microbiome following ingestion of soy isoflavone daidzein. Having established and replicated proof- of-concept (preliminary studies), we will assess the in vivo efficacy of S-equol to delay the progression to neurocognitive decline; synaptodendritic complexity, neuroinflammation and oxidative stress will be assessed in different time-sequential cohorts as the basis of the preserved neurocognitive function. Initially we will establish the optimal 2-month therapeutic window. Subsequently, we will establish the functional role of alterations in synaptodendritic complexity, neuroinflammation and oxidative stress as neurobiological mechanisms contributing to, if not mediating, the neurocognitive impairments consequent to chronic expression of the HIV-1 transgene. The program goal is to advance the field by establishing 1) the progression of neurocognitive decline in a translationally relevant model of HAND, 2) the efficacy of a regenerative approach targeted at synaptodendritic complexity in protecting (slowing or halting) the neurocognitive decline in the core cognitive components of attention and executive function, and 3) proof-of- principle for the microbiota-gut-brain axis as an innovative therapeutic approach for HAND.

Project Summary: Apathy is a common motivational alteration in HIV-1+ individuals, affecting between 30-60% of the population, despite antiretroviral therapy. The high prevalence of apathy in HIV-1+ individuals is one of the biggest challenges facing HIV clinicians and researchers alike; there is a critical need to develop treatment approaches for HAND. Using the HIV-1 transgenic (Tg) rat model in a prospective longitudinal design, we will test the hypothesis that the development and progression of dysregulation of motivational processes associated with HIV-1 are consequent to synaptodendritic pathology, neuroinflammation and oxidative stress of the dopamine (DA) system in the prefrontal cortex-basal ganglia axis, and that the trajectory of motivational dysregulation will be altered with a therapeutic approach targeted to the mesocorticolimbic DA system. The specific aims are: 1) To establish the development and progression of motivational dysregulation in the HIV-1 Tg rat as measured with drug, food, and natural reinforcers, as distinct from potential changes in sensory and activity confounds. The progression of neurocognitive dysfunction will be assessed with periodic tests from pre-adolescence through advanced age, given the exponential growth of 65+ year old HIV+ patients. Both males and females will be examined. 2) To establish the progression of CNS synaptodendritic pathology in the prefrontal cortex (PFC) and medium spiny neurons (MSN) of the nucleus accumbens of the HIV-1 Tg rat with a focus on quantifying dendritic branching and spine parameter alterations. Specifically, synaptodendritic complexity, neuroinflammation, and oxidative stress will be assessed in different cohorts as the basis of the dysregulation of motivational processes. 3) To establish the treatment of the dysregulation of motivational processes consequent to chronic, low-level inflammation and HIV-1 protein exposure with currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction. With currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction we will mechanistically test the functional role of the neurobiological changes in the DA receptors and dopamine transporter (DAT) as a neurochemical mechanism contributing to, if not mediating, the motivational dysregulation consequent to chronic expression of the HIV-1 transgene. Initially we will establish the optimal therapeutic window. These studies will establish the functional role of alterations in synapto- dendritic complexity, neuroinflammation and oxidative stress as neurobiological mechanisms contributing to, if not mediating, the dysregulation of motivational systems consequent to chronic expression of the HIV-1 transgene. The program goal of our longitudinal studies is to establish our ability to predict motivational dysregulation as a function of chronic expression of the HIV-1 transgene, to provide a greater mechanistic understanding of the interrelationships of HIV-1-induced apathy and DA system dysfunction, and to improve the therapeutic options relevant to HIV-1+ individuals and improve their quality of life.