Frances M. Leslie - US grants

The morphological changes which accompany development of the nervous system have been well characterized. In contrast, the neurochemical and molecular events which underlie these processes are less well understood. We propose to utilize a combination of neuroanatomical and biochemical techniques to elucidate several aspects of the functional role of opioid systems during development of the CNS. The differential developmental appearance of opioid receptor subtypes will be characterized, and will be correlated with the appearance of specific opioid peptide systems. The pharma-ological characteristics of these early receptors will be analyzed and compared to those of the adult. The hypothesis that Mu and Delta receptor subtypes represent intercoverting forms of the same receptor will also be examined in detail. Well characterized behavioral and neuroendocrine deficits have been shown to result from prenatal exposure to morphine. We propose to analyze the molecular basis of these deficits, by comparing several aspects of hypothalamic organization in normal and drug-treated animals. This analysis should provide data relevant to the hypothesis that opioid receptors are important in guidance of neuronal migration.

The role of neuropeptides in the complex developmental processes which underlie the morphogenesis of mammalian brain is currently unknown. Opioid peptides and their receptors appear early in ontogeny, being detectable within embryonic rat brain by the fourteenth day of gestation. Data from both clinical and animal studies suggest that opioid receptors may possess some functional capacity at an early stage of neurogenesis. Our goal is to use a combination of neuroanatomical and biochemical techniques to elucidate several aspects of the ontogeny, and possible developmental role, of opioid systems within the CNS. Autoradiographic techniques will be used to continue our detailed studies of the development of opioid receptors within rat brain. We shall undertake both quantitative and qualitative analyses of the developmental appearance of Beta-endorphin binding sites. In a parallel study, caudate putamen will be used as a model in which to compare the detailed temporal and spatial relationships between opioid peptide and receptor development. Further experiments, in intact animals, are designed to identify possible developmental roles of endogenous opioid systems in the control of cellular proliferation and migration. Both quantitative autoradiographic and biochemical techniques will be used to further characterize the effects of exogenous and endogenous opioids on DNA synthesis within neonatal rat brain. Combined (3H) thymidine autoradiographic and immunohistochemical techniques will be used to characterize the "birthdate" of a recently identified population of Beta-endorphin immunoreactive cells which appear transiently within subventricular germinal zone. Immunohistochemical co-localization techniques will be used for a more detailed examination of the effects of opioids on the growth and survival of developing neural cells. Opioid actions will be examined, both in untreated cultures, and in those which have been stimulated by growth factors. The effects of growth factors on the development of opioid systems in culture will also be determined. These studies are important in that they may provide some insight as to the organizational principles which underlie neuronal development within mammalian CNS, as well as to possible consequences, to the offspring, of maternal drug abuse.

Previous studies of cortical development have demonstrated dynamic patterns of neurotransmitter innervation, which may have functional significance in directing the complex processes of cortical ontogeny. Although acetylcholine (ACh), in particular, has been implicated as a modulator of cortical ontogeny, little is known about the ontogenetic appearance of the receptors through which this system mediates its biological effects. Thus, a detailed characterization of the developmental patterns of cholinergic receptor appearance is essential. By using techniques which permit analysis of the anatomical localization of receptors, cholinoceptive elements within cortex may be identified, and the mechanisms by which ACh influences cortical structure and function may be further elucidated. The aim of the present proposal is to examine the developmental appearance and functional role of cholinergic receptors in rodent somatosensory cortex. Using receptor autoradiography, in situ hybridization and immunohistochemistry, the laminar and regional distribution of receptors will be examined in somatosensory cortex of rats of defined pre- and post- natal ages. The developmental appearance of cholinergic receptors will be analyzed both within and across individual cortical layers, and will be compared with that of thalamocortical afferents. Particular attention will be paid to the first ten postnatal days, in order to identify those receptors which may modulate crucial aspects of cortical development. Of particular interest will be those receptors which exhibit either a transient appearance and/or a distribution which corresponds to some aspect of the somatotopic map, particularly the whisker barrel field. In order to more accurately predict the possible functional role of such receptors, we shall undertake a detailed examination of their cellular localization. Using quantitative autoradiography, receptor distributions will be examined at appropriate postnatal ages in animals which have received lesions of thalamocortical or serotonergic afferents, or of intrinsic cortical neurons. The timecourse of receptor loss, if any, will be compared with that of cellular loss, as determined by histochemical or autoradiographic techniques. Having identified those receptors which are localized on either thalamocortical afferents or cortical neurons, we shall examine the consequences of their selective pharmacological manipulation on cortical development. We shall first determine whether the receptors of interest are expressed in organotypically cultured thalamocortical slices, and then examine the effects of selective drug treatments on the development of cortical neurons and their thalamocortical innervation in vitro. This will permit analysis of drug effects under carefully controlled experimental conditions. Overall, these studies should provide insight into, not only the cellular localization and physiological role of cholinergic receptors in developing cortex, but also the consequences of their manipulation by the exogenous administration of clinically relevant drugs.

In the previous funding period, we have demonstrated that nicotine interacts directly with nicotinic receptors (nAChRs) on catecholamine neurons to stimulate norepinephrine (NE) and dopamine (DA) release in developing rat brain. We now propose a series of studies to examine the physiological and clinical relevance of these observations. We will study the species specificity of nAChR-mediated catecholamine release in C57BL/6J mice, and will use null mutant mouse strains to examine the subunit composition of nAChRs on developing NE and DA terminals. Two types of approach will be used to examine whether nAChRs on developing noradrenergic locus coeruleus (LC) terminals may be activated by endogenous ligand. We will use immunohistochemistry to establish the developmental appearance of cholinergic cells and fibers in relationship to LC and its forebrain and hindbrain terminal fields. Antibodies specific for the vesicular acetylcholine transporter (VAChT) and for dopamine beta hydroxylase (DBH) will be used to visualize developing cholinergic and noradrenergic elements, respectively. We will also determine whether nAChR activation or blockade can modify early learning in paradigms that have been shown to require activation of the LC, including early olfactory learning and somatosensory associative conditioning. Learning in each task has been shown to necessitate LC activation by tactile stimulation and electrical shock, respectively. We will test the hypothesis that peripheral administration of nicotine induces sufficient LC stimulation to facilitate learning in the absence of sensory stimuli. We will also use the nAChR antagonist, mecamylamine, to determine whether endogenous acetylcholine (ACh) influences sensory stimulus-induced LC activation on these tasks. Having established the functional roles of nAChRs in acute regulation of catecholamine release, we will examine the effects of both tonic and phasic modes of chronic prenatal nicotine exposure on the development of central catecholamine systems. Osmotic minipumps will be used for constant infusion and self- administration for pulsatile delivery of nicotine to pregnant rats. Offspring of varying ages will be used for functional and neuroanatomical studies to determine whether prenatal nicotine exposure after the overall excitability, or nicotine-sensitivity, of NE and DA terminal fields. Behavioral tests that involve both central NE (early olfactory learning, associative somatosensory conditioning) and DA systems (locomotion, conditioned place preference, self-administration) will be used to determine whether chronic nicotine-induced changes in catecholamine system development are associated with long-term alterations in behavioral response. These combined in vitro and in vivo approaches are designed to elucidate mechanisms underlying adaptive responses of central catecholamine neurons to chronic prenatal nicotine exposure.

The majority of adult smokers initiate smoking as children. Most previous studies of the biological effects of nicotine have focused on adults rather on adolescence, a developmental period that is characterized by sexual maturation and by significant changes in brain structure and function. In the present proposal we will use animal models to examine the biological factors that underlie susceptibility to nicotine. In particular, we will test the following hypotheses: (l) That the balance of reinforcing/aversive effects of nicotine is greater in juveniles than in adults. (2) That individual differences in susceptibility to nicotine dependence are associated with variations in stress responsivity. Three specific aims are proposed. First, the effects of nicotine will be compared in juvenile rats aged postnatal day (p) 30 and in adults. Animals will be treated with nicotine and tested behaviorally. At the end Of some experiments animals will be decapitated and trunk blood collected for the measurement of corticosterone levels. Brains will be processed by in situ hybridization for quantitative analysis of the expression of mRNA for the immediate early gene, cfos, in specific neuronal populations. Experiments will be conducted with both male and female rats to determine whether nicotine is more reinforcing in juveniles than adults using both conditioned place preference and self adrnistration paradigms. Animals will also be tested to determine whether conditioned sensitization to the locomotor activating effects of nicotine Occurs more rapidly in juveniles than adults, and to determine whether juvenile exposure to nicotine increases sensitivity to nicotine in adults. Second, individual differences in stress responsivity will be induced by a procedure in which pups are either handled (H) or not handled (NH) for two weeks after birth. H and NH groups will then be compared as juveniles and adults to determine whether differences in stress responsivity result in different acute effects of nicotine on locomotion, plasma corticosterone and cfos expression. Differences in the reinforcing effects of nicotine in H and NH groups will also be examined, as will differences in withdrawal severity following chronic nicotine treatment. Third, the effects of nicotine on genetically distinct strains of mice will be studied. Both acute and chronic effects of nicotine in C3H andC57B1/6J mice, and in transgenic mice with alterations in selected genes, will be examined. These studies will provide critical information on the biological mechanisms underlying individual variations in responsivity to nicotine and will complement the human studies in projects I and 2.

The proposed TTURC reflects several unique strengths. First, the Center brings together five eminent project leaders with diverse expertise in the field of tobacco research (Drs. Leslie, Janner, PotInn, Pechman, and Tengs). Each of these scholars has achieved distinction in their respective disciplines of pharmacology, neuroscience, human development, social and health psychology, community health promotion, and health policy analysis. These scientists will work collaboratively to identify key predictor of nicotine dependency in animals and tobacco susceptibility and use in humans. A major goal of the collaboration is to establish a shared conceptual model of tobacco susceptibility and use that can be used to integrate experimental findings across multiple levels of analysis (i.e., biological, behavioral, developmental, interpersonal, and epidemiological/community levels), and to translate these findings into intervention strategics (i.e., pharmacological, behavioral, social marketing, and health policy strategies) that will be evaluated for their public health efficacy and cost effectiveness. A second major strength of the TTURC is its incorporation of four highly interactive cores, each strategically designed to promote successful transdisciplinary collaboration in the field of tobacco susceptibility, use, and intervention. The Transdisciplinary Core will implement structured activities to foster scientific collaboration (e.g., team workshops, retreats), and also conduct a systematic self study of collaboration among members of the TIURC over a five-year period, based on a taxonomic model of the antecedents, intermediate processes, and scientific outcomes of transdisciplinary research. The Informatics Core (led by Dr. Shankle) will develop several valuable informational resources, including a shared relational data base to facilitate the integration of data sets and research findings across multiple TTURC projects and an interactive TTURC web site), all designed to promote more effective transdisciplinary collaboration The Career Development Core (co-directed by Drs. Whitely and Fallon) integrates the theoretical and methodological perspectives emphasized in the research projects and the Transdisciplinary, and Informatics Cores. It will afford a wide array of transdisciplinary training opportunities that are directly responsive to the training priorities outlined in the Report of the Working Groups of the Youth Tobacco Prevention Initiative (1998). Finally, the Administrative Core will be co-directed by Drs. Leslie and Stokols, both of whom have extensive university administrative experience and are active in the fields of pharmacology, neuro5cience, community health promotion, social ecology, and transdisciplinary theory development. The CO-PIs will oversee interactive activities of the TTURC with the goal of promoting effective transdisciplinary collaboration among the project team members research trainees and staff and members of the External and Internal Advisory Boards.

DESCRIPTION (provided by applicant): Adolescence is a critical period of vulnerability for the onset of substance abuse. Those who begin drug use in early adolescence show a pattern of heavier lifetime consumption and greater difficulty quitting than those who start as older adolescents or young adults. Epidemiological studies have characterized a progression of drug use from alcohol and tobacco to marijuana and other illicit drugs. Such findings have led to the hypothesis that alcohol and tobacco may serve as 'gateway' drugs that sensitize reward pathways to the action of illicit drugs. We propose to undertake integrative behavioral and neurochemical/neuroanatomical studies in rats to evaluate this hypothesis. In Specific Aim 1, we will use intravenous self-administration to determine whether there are age and sex differences in the rewarding effects of cocaine and amphetamine. Acquisition tests will be conducted for each drug during early adolescence (postnatal day (P) 28), late adolescence (P38) and adulthood (P90). In separate groups of animals, the effects of acute and chronic administration of drug on locomotor activity will be examined at each age. The brains of these animals will then be processed by in situ hybridization for analysis of drug-induced neuronal activation, as measured by expression of mRNA for the immediate early gene c-fos. In Specific Aim 2, we will conduct studies to evaluate whether nicotine increases the sensitivity of adolescent and/or adult brain to the actions of amphetamine and cocaine. Self-administration studies will be undertaken to determine whether acute, non-contingent nicotine treatment can increase the reinforcing value of cocaine or amphetamine in adolescents or adults. The effect of chronic, intermittent daily injection of nicotine on subsequent acquisition of cocaine and amphetamine self-administration will also be examined. In parallel studies, the effect of chronic nicotine on psychostimulant-induced locomotor activity and neuronal activation will also be studied in adolescents and adults. Although adolescence is the principal timeframe for initiation of drug use, few studies have evaluated mechanisms of drug action at this developmental timepoint. The combined behavioral and neuroanatomical studies of psychostimulant action that we propose should provide critical understanding of the actions of abused drugs on adolescent brain, and clarify whether these are different or similar to those in adult.

[unreadable] DESCRIPTION (provided by applicant): Although current research into the causes of smoking focuses largely on nicotine, there is accumulating evidence that other tobacco constituents, such as monoamine oxidase (MAO) inhibitors, may increase the addictive liability of tobacco use. The goal of the proposed research is to elucidate the contribution of MAO inhibition to the psychoactive effects of tobacco. We will use the rat as a model to test the hypothesis that MAO inhibition enhances the effects of nicotine on smoking initiation and dependence. We base this hypothesis on prior findings that i) MAO is an important modulator of monoamines in brain reward systems, ii) smoking reduces brain MAO activity thereby increasing brain monoamines, and iii) MAO inhibition enhances nicotine-induced locomotor sensitization and reward. We propose to explore the effects of MAO inhibition on nicotine-induced actions at the behavioral, biochemical and anatomical levels. The specific aims are: 1. To determine the selectivity requirements for MAO inhibitor enhancement of nicotine's reinforcing actions. We will use adult male and female rats to evaluate how the selectivity of MAO inhibitors affects the acquisition of nicotine self-administration; 2. To determine how MAO inhibition alters nicotine- induced changes in regional brain activity. We will use neuroanatomical and neurochemical approaches to evaluate the neural circuitry underlying the enhancement of nicotine reward by the MAO inhibitor, tranylcypromine; 3. To determine whether tranylcypromine selectively enhances nicotine reward as compared to other psychostimulants. We will compare the effect of tranylcypromine pretreatment on the acquisition of nicotine, cocaine and amphetamine self-administration. If selectivity for nicotine is observed, we will test whether MAO inhibition alters nicotine's relative reward strength in a 2-lever choice paradigm; 4. To determine if MAO inhibition increases nicotine withdrawal as a measure of dependence. Animals which are chronically infused with nicotine will be treated with saline or tranylcypromine and compared for mecamylamine- and naloxone-precipitated withdrawal symptoms and conditioned place aversion. The results of these studies should advance our understanding of the interaction of nicotine with other tobacco constituents, and lead to improved animal models of smoking that will strengthen our search for tobacco addiction's causes and cures. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Tobacco use remains a major cause of premature death in the United States, with 30% of cancer deaths and 18% of all deaths directly attributable to smoking. Initiation of tobacco use occurs primarily during adolescence, with smoking rates peaking in young adults. Although tobacco smoke contains over 8000 constituents, most animal models used for testing smoking cessation therapies focus on the effects of nicotine alone in adult males. This lack of attention to both the non-nicotine constituents of tobacco and the age of first use may account for the low success rate of current tobacco cessation medications. With clinical data showing sex differences in tobacco dependence, it is also important to study females. Our group has been one of the only ones to examine the impact of non-nicotine constituents on nicotine actions in both adolescents and adults. We have shown that adult and adolescent rats will self-administer cigarette smoke extract (CSE), made by bubbling cigarette smoke through saline solution. CSE is more reinforcing in adult male rats than equivalent doses of nicotine alone, and animals that self-administered CSE are more sensitive to both stress- and nicotine-induced reinstatement of drug-seeking behavior than animals that worked for nicotine alone. Our data also suggest that both adolescents and adults exhibit more somatic withdrawal upon cessation of chronic CSE administration than nicotine. These findings indicate the importance of assessing the effects of nicotine in the presence of other tobacco smoke constituents. The objective of the current application is to examine mechanisms underlying enhanced withdrawal and drug-seeking behavior in animals that have been chronically treated with CSE. In addition to evaluating adolescent and adult males, we will initiate one of the first studies of the impact of CSE exposure in females. The specific aims of the proposal include: 1) Comparison of somatic and affective withdrawal symptoms following chronic nicotine and CSE exposure in adolescent and adult rats of both sexes. We will further test the hypotheses that monoamine oxidase inhibition, enhanced nicotinic acetylcholine and/or kappa opioid receptor function underlie the increased somatic withdrawal that we have observed following cessation of chronic CSE treatment in adult and adolescent males; 2) Comparison of stress-induced reinstatement of nicotine- and CSE-seeking behavior in adolescent and adult males, and assessment of the role of a3ß4 nicotinic receptors in mediating drug craving; 3) Establish a dose-response relationship for acquisition of self-administration of CSE, and equivalent doses of nicotine alone, in females. The approach is innovative in that it aims to bridge the gap between human and animal studies by examining the combined impact of tobacco smoke aqueous components. The proposed research is significant in that it provides an initial step towards a more comprehensive approach to determining neural mechanisms underlying tobacco dependence, and may result in the development of improved clinical therapies for smoking cessation.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.