Steven R. Childers, Ph.D. - US grants

DESCRIPTION: (Applicant's Abstract) Opioids, including drugs like morphine and heroin as well as endogenous opioid peptides, bind to receptors that belong to the superfamily of G-protein-coupled receptors. The best studied actions of opioids are mediated through the Gi/o family of transducers, where the effectors include inhibition of adenylyl cyclase, activation of potassium conductance and inhibition of calcium channels. Since its inception, the overall goal of this project has been to characterize the G-protein-coupled signal transduction pathways for opioid receptors in brain. During the past funding period, this goal was significantly aided by the development of agonist-stimulated [35S]GTPgS binding as a measure of receptor activation of transducers. These studies showed that the neuroanatomical resolution of this method could be dramatically extended with in vitro [35S]GTPgS autoradiography in brain sections. This development allowed us to address questions which cannot be addressed by any other technique, and explore the regional specificity of agonist efficacy. In the future planned studies for this project, this technique will be combined with other approaches to provide a molecular understanding of mechanisms of opioid signal transduction in neurons. First, the concept that opioid agonist efficacy varies in different brain regions will be tested with a combination of full and partial agonists assayed for maximal efficacy in stimulating [35S]GTPgS binding and in receptor/transducer amplification. Second, novel GTP analogs and procedures will be developed to extend the method of [35S]GTPgS binding to allow for increased anatomical resolution and to determine G-protein subtypes coupled to opioid receptor types in different regions. Third, [35S]GTPgS autoradiography will be performed in animal models such as opioid receptor transgenic knockout mice to determine whether these genetic manipulations have altered specific receptor-G-protein coupling in different brain areas. Fourth, the concepts learned by assay of [35S]GTPgS binding will be extended to a downstream target of the opioid receptor second messenger system: forskolin-stimulated expression of pro-enkephalin mRNA in mu receptor-transfected C-6 glioma cells. Finally, mechanisms of chronic opioid action on receptor-G-protein interactions will be examined in both brain sections and in mu receptor-transfected NG108-15 cells.

Opiates exert actions by binding to specific receptors, whose interactions are translated across the cell membrane into specific biological responses by second messenger systems. A variety of data suggest that opiate tolerance may be associated with a step distal from the receptor binding site. Therefore, a logical candidate for the molecular mechanism of tolerance is coupling of opiate receptors with second messenger systems. The principal second messenger system associated with opiate receptors thus far has been opiate-mediated reductions in cellular cyclic AMP levels through inhibition of adenylate cyclase. However, other second messenger systems are also possibly coupled to opiate receptors. Preliminary data in cultures of C-6 glioma cells has revealed the existence of another such system: opiate-stimulated adenylate cyclase. In these cells, opiate agonists both inhibit and stimulate adenylate cyclase, and both activities are blocked by naloxone. Each activity can be studied separately by selective blockade of the other activity: pertussis toxin to block inhibited activity, and pretreatment of membranes at pH 4.5 to block stimulated activity. This proposal will explore the specificity of opiate-stimulated and -inhibited adenylate cyclase in C-6 glioma cells, and determine whether different receptor subtypes are responsible for these two activities. Because these two activities are opposite, they may mask each other in many different cell types. Therefore, other cell types in addition to C-6 glioma cells will be screened for either opiate-stimulated or -inhibited adenylate cyclase with the same techniques. The control of the balance between stimulated and inhibited activities will be studied by fusion of membranes from different cell types with different activities. Finally, the effects of chronic opiate exposure on these systems will be examined by treatment of cells in culture with different agonists. The goal of these experiments is to determine whether these model cell systems can reveal important information about the role of opiate receptor-coupled second messenger systems in the development of opiate tolerance.

Opiates and opioid peptides act by binding to specific receptor binding sites which are coupled to second messenger systems in neuronal cell membranes. One such second messenger system is opiate-mediated inhibition of adenylate cyclase, in which receptors are coupled to adenylate cyclase through specific auanine nucleotide binding proteins. This activity can now be measured reproducibly in brain membranes by a technique of low pH pretreatment which selectively eliminates stimulation of adenylate cyclase by other receptors but increases inhibition of adenylate cyclase by opiate agonists. This technique also increases the regulation of opite agonist binding sites by guanine nucleotides. This project will study the molecular mechanisms in brain membranes that regulate the coupling of opiate receptor to adenylate cyclase. First, it will explore the pharmacological properties of opiate-inhibited adenylate cyclase and determine the nature of the opiate receptor subtypes involved in this second messenger system. Second, this research will study the biochemical factors which regulate the activity of this system: by labeling the guanine nucleotide binding proteiins in brain membranes, it will determine the nature of the low pH effect which alters the balance between stimulated and inhibited adenylate cyclase. Finally, the project will focus on the effects of opiate tolerance on this second messenger system. Opiate tolerane does not appear to involve a loss of opiate receptor binding sites; however, it may involve a loss of coupling between receptors and adenylate cyclase. These studies will examine this possibililty and search for possible biochemical changes in quanine nucleotide binding proteins that may occur in response to tolerane. This research will not only help describe some of the molecular mechanisms of tolerance in brain, but will also help to explain acute actions of opiates by exploring the factors in brain membranes that regulate expression of opiate-inhibited adenylate cyclase.

endogenous opioid; receptor coupling; G protein; drug tolerance; drug addiction; second messengers; opioid receptor; phosphorylation; neuropharmacology; stimulant /agonist; morphine; biological signal transduction; corpus striatum; self medication; inhibitor /antagonist; drug screening /evaluation; synapses; messenger RNA; reinforcer; psychopharmacology; receptor sensitivity; laboratory rat;

The cannabinoids have a long history as drugs of abuse as the active psychoactive ingredients in marijuana. Nevertheless, the molecular mechanisms of action of these drugs are still not clear. A crucial breakthrough in this field occurred with the discovery that delta9- tetrahydrocannabinol (THC) and its analogs bind to brain membrane receptors which are coupled to G-proteins to inhibit adenylyl cyclase. While these cannabinoid receptors may not be responsible for all CNS actions of cannabinoids, they explain many of the specific neuronal actions of these compounds. Moreover, since many other neurotransmitters and neuromodulators bind to G-protein-coupled receptors, these results suggest that cannabinoids may not simple be exogenous drugs of abuse but also be members of a novel class of neurotransmitters. The current study will explore components of the putative endogenous cannabinoid system in the brain by utilizing both traditional cannabinoid ligands as well as a novel class of cannabinoid ligands known as the aminoalkylindoles. These receptors can be studied by radioreceptor binding to brain membranes, by GTP-dependent inhibition of adenylyl cyclase in cerebellar membranes and cerebellar granule cells, by stimulation of low k/m GTPase in cerebellar membranes, and by inhibition of electrically-induced contractions of mouse vas deferens. This project will focus on cannabinoid actions in cultured cerebellar granule cells, which represent a novel, non-transformed cell culture system for the study of cannabinoid receptors. In the cerebellar granule cells, the effects of cannabinoids on glutamate release will be explored, in order to demonstrate that these compounds act like other inhibitory agonists in cerebellum to inhibit glutamate release. Specific antibody and antisense experiments will be utilized to identify the specific G-proteins coupled to cannabinoid receptors in different areas of the brain. A major component of this project will continue to characterize endogenous cannabinoid ligands from brain extracts. The previous grant period isolated one such compound from bovine brain. Although its structure is not yet known, many of its chromatographic properties suggest that it is structurally different from anandamide, an ethanolamine derivative of arachidonic acid recently identified as a potential endogenous cannabinoid. These studies will determine the structure and localization of this novel cannabinoid liqand, and compare these properties to those of other compounds isolated by other laboratories.

Many neurotransmitters in brain, as well as several drugs of abuse, act by binding to receptors belonging to the superfamily of G-protein-coupled receptors. These drugs include the opioids, which act directly at their own G-protein receptors, and cocaine, which increases extra-synaptic dopamine that binds to its own receptor. The goal of this project is to utilize animal models provided by the Center to examine how chronic treatment with opioids and psychostimulants affect coupling of different receptors to G-proteins in brain, and how these changes at the level of the receptor amd transducer then translate into downstream changes in opioid-mediated second messenger systems. In the first case, the coupling of receptor to G-proteins will be explored by [35/S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for a number of different receptors to be assayed simultaneously in brain sections from the same animals, this provides an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies which showed that chronic morphine treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. These studies will determine the time course of the chronic morphine effect, and compare chronic treatment of rats with morphine to chronic treatment with other opioid agonists of different potencies and efficacies. The effects on non-contingent chronic administration morphine. To determine how these changes in receptor-G- protein coupling affect opioid second messenger systems, rats treated in the same way will be analyzed for opioid inhibition of forskolin- stimulated pro-enkephalin signal transduction systems in vivo. A second aim will examine the effect of chronic administration of cocaine and other psychostimulants on receptor-coupled G-proteins, with a particular focus on D2 dopamine receptors, 5-HT/1A receptors, and opioid receptors. To determine the effects of transporter selectivity and pharmacokinetics on modulation of receptor coupling to G-proteins, novel tropanes synthesized by the Center with well-defined specificities and durations of action will be administered chronically. This project will use information obtained from other Center projects in the planning of its studies, and provide information about receptor changes during chronic drug treatment that will be important for studies in other projects.

The Center for the Neurobiological Investigations of Drug Abuse (CNIDA) was established in 1991 by a grant from NIDA to support an integrated research program in the neuroscience of drug abuse at Bowman Gray School of Medicine at Wake Forest University. The Specific Aims of the Center are: to provide an environment to promote collaborative and multi- disciplinary research into the neurobiological mechanisms of drug abuse among scientists at Bowman Gray School of Medicine; to provide a focus for the training of students and post-doctoral fellows in contemporary methods for investigation of the neurobiological basis of drug abuse; and to serve as an information source to both the lay and scientific community on issues related to the neurobiological aspects of drug abuse and to serve as the scientific reference for treatment and prevention programs in the community. The Center will accomplish these goals though the activities of three cores and eight projects over this next funding period. The cores Administration, Animal and Chemistry will provide services to the project that include an interdisciplinary research approach that spans from the molecular biology to non-human primate self-administration. Seventeen faculty will directly participate in Center research that includes individuals from the Departments of Physiology-Pharmacology and Radiology at Bowman Gray School of Medicine, and the Department of Chemistry at SUNY-Buffalo with expertise in receptor mechanisms, synthetic organic chemistry, medicinal chemistry, radiochemistry, neuroanatomy, positron emission tomography, neurochemistry, neuropharmacology, molecular biology, neurophysiology and rodent and primate behavioral pharmacology. These individuals will work together on CNIDA research projects directed toward a further understanding of the neuroscience of drug abuse. The Center will continue to provide research training for undergraduates, graduate students and postdoctoral fellow and serve as a resource on the physiology and neurobiology of drug abuse in scientific and lay communities.

Opioid agonists like morphine, codeine and meperidine remain the most commonly used and effective treatment for chronic pain conditions. Despite the fact that chronic opioid treatment can produce high levels of tolerance and physical dependence. Mechanisms of tolerance and dependence for in brain are not well understood, but it is clear that chronic opioid treatment produces significant receptor desensitization in specific brain regions. Moreover, pain itself, as well as concurrent treatment with drugs like adenosine and alpha2- adrenergic agonists, alter the sensitivity of patients to opioid treatment. Opioid receptors (including mu, delta and kappa types), as well as adenosine A1 and alpha2-adrenergic receptors, are G-protein-coupled receptors, and their ability to activate signal transduction systems can be determined by the ability of agonists to stimulate [35S]GTPgammaS binding in both membranes and section autoradiography. This project will examine regulation of several receptor/G-protein interactions in rat spinal cord, using models of drug treatment, self-administration and chronic pain developed by other Center components. First, after determining the acute efficacies of opioid and adenosine A1 agonists in activating G-proteins in spinal cord, the ability of chronic drug exposure to produce receptor desensitization will be examined in both spinal cord membranes and by autoradiography. These treatments will include chronic intrathecal opioid administration to desensitize opioid receptor-activated G-proteins, and chronic intrathecal administration of adenosine and clonidine to desensitize agonist-stimulated incorporation of [32P]AAGTP into specific G-protein subunits. Second, various receptor-activated G-protein activities will be determined in both brains ans spinal cords from spinal nerve ligated rats to determine whether chronic pain and hypersensitivity affect receptor/G-protein coupling. These studies will also determine how chronic pain states modulate basal levels and activities of G-proteins in spinal cord. Third, the ability of NMDA antagonists and protein kinase C inhibitors to modulate receptor desensitization will be tested after chronic intrathecal administration of drugs. Information from this project will help design studies in the Clinical Core to test the use of opioid agonists of differing efficacies in treating chronic pain. Moreover, these studies will provide information to minimize tolerance in long-term drug treatment of chronic pain.

DESCRIPTION (provided by applicant): The Center for the Neurobiological Investigation of Drug Abuse (CNIDA) was established in 1991 by a grant from NIDA. The Specific Aims of the Center are: to provide an environment to promote collaborative and multidisciplinary research into the neurobiological mechanisms of drug abuse among scientists at Wake Forest University School of Medicine; to provide a focus for the training of students and postdoctoral fellows in contemporary methods for investigation of the neurobiological basis of drug abuse; and to serve as an information source to both the lay and scientific community on issues related to the neurobiological aspects of drug abuse and to serve as the scientific reference for treatment and prevention programs in the community. The Center will accomplish these goals through the activities of four cores and seven projects over this next funding period. The cores (Administration, Animal, Chemistry and PET Imaging) will provide services to the projects whose expertise spans from molecular biology to non-human primate self-administration. The projects are diverse, but their research encompasses a general theme to examine the neuronal adaptations occurring in brain as a result of drug use, and which mediate the transition from drug use to drug addiction. These projects utilize several animal models and technologies uniquely developed by the Center investigators. A large number (23) of faculty and research associates will directly participate in Center research, from the Departments of Physiology-Pharmacology and Radiology at Wake Forest University School of Medicine, as well as the Department of Chemistry at SUNY-Buffalo. The Center will continue to provide research training for undergraduates, graduate students and postdoctoral fellows and serve as a resource on the neurobiology of drug abuse to the scientific and lay communities.

Psychostimulants, including cocaine as well as novel cocaine analogs developed by this Center, act by blocking transporter-mediated reuptake of biogenic amines (dopamine, 5-HT and norepinephrine). This project utilizes novel tropane and methylphenidate analogs, along with specific animals models developed by other Center projects, to study transporter binding sites, and to examine the consequences of chronic drug treatment on G-protein-coupled receptors. The first aim of this project utilizes animal models developed by the Center to examine how chronic treatment with psychostimulants (both cocaine and novel cocaine analogs) affect coupling of biogenic amine receptors to G-proteins in brain. These studies will utilize [35S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for an number of different receptors to be assayed simultaneously in brain sections from the same animal, it is an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies showing that chronic opioid treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. Three different drug treatment paradigms will be employed: a chronic treatment with the highly potent tropane analog WF-23, chronic self-administration in a binge model of cocaine developed by Dr. Roberts in subproject 0011, and a speedball (heroin/cocaine combination) self-administration model developed by Drs. Smith and Martin in subproject 0005. The second aim will characterize the properties of novel irreversible cocaine analogs (both tropanes and methylphenidates) as probes of dopamine transporter structure and function. Since these compounds will be used in behavioral studies by other Center projects, our laboratory will first characterize the basic irreversible binding properties of these analogs both in vitro and in vivo, using transporter radioligand binding in membranes and autoradiography. The most potent analogs will be labeled with [1251] and differences in binding between tropanes and methylphenidates will be examined by SDS-PAGE and purification of [1251]-labeled tryptic peptide fragments isolated from cells transfected with dopamine transporters.

Psychostimulants, including cocaine as well as novel cocaine analogs developed by this Center, act by blocking transporter-mediated reuptake of biogenic amines (dopamine, 5-HT and norepinephrine). This project utilizes novel tropane and methylphenidate analogs, along with specific animals models developed by other Center projects, to study transporter binding sites, and to examine the consequences of chronic drug treatment on G-protein-coupled receptors. The first aim of this project utilizes animal models developed by the Center to examine how chronic treatment with psychostimulants (both cocaine and novel cocaine analogs) affect coupling of biogenic amine receptors to G-proteins in brain. These studies will utilize [35S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for an number of different receptors to be assayed simultaneously in brain sections from the same animal, it is an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies showing that chronic opioid treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. Three different drug treatment paradigms will be employed: a chronic treatment with the highly potent tropane analog WF-23, chronic self-administration in a binge model of cocaine developed by Dr. Roberts in subproject 0011, and a speedball (heroin/cocaine combination) self-administration model developed by Drs. Smith and Martin in subproject 0005. The second aim will characterize the properties of novel irreversible cocaine analogs (both tropanes and methylphenidates) as probes of dopamine transporter structure and function. Since these compounds will be used in behavioral studies by other Center projects, our laboratory will first characterize the basic irreversible binding properties of these analogs both in vitro and in vivo, using transporter radioligand binding in membranes and autoradiography. The most potent analogs will be labeled with [1251] and differences in binding between tropanes and methylphenidates will be examined by SDS-PAGE and purification of [1251]-labeled tryptic peptide fragments isolated from cells transfected with dopamine transporters.

The Center for the Neurobiology of Addiction Treatment (CNAT) is a new version of a PSO Center at Wake Forest University Health Sciences that has been continuously funded by NIDA since 1991. The Specific Aims are: 1. to provide a novel mechanism of interaction between NIDA Investigators at Wake Forest University to examine neurobiological mechanisms of cocaine pharmacotherapies, using tightly interacting projects to ensure an effective collaborative structure;2. to provide a formal structure of collaborative interactions between our Center and our clinical partners at two NIDA-funded clinical centers, thus providing a novel translational component to our research;3. to provide a focus for the training of students and postdoctoral fellows in contemporary methods for investigating the neurobiological basis of drug abuse;4. to serve as an information source to both the lay and scientific community on issues related to the neurobiological aspects of drug abuse. The scientific theme of the Center is focused on examining the neurobiological mechanisms of action of drugs that are currently used, or proposed for use in the clinic for treatment of cocaine addiction;these studies will begin with topiramate, and proceed with other potential treatment agents including modafinil, D3 dopamine antagonists, orexin antagonists, and others. The Center will accomplish these goals through the activities of two cores and three major projects. The projects are designed to be highly interactive, with each project's activities dependent on results obtained from the other two projects. Project 1 will examine effects of drug treatment on various behavioral models in both rats and monkeys;Project 2 will explore how these drug treatment paradigms affect functional consequences in brain through neuro-imaging and voltammetry;and Project 3 will explore how these drug treatments affect gene and protein expression, receptor function and signal transduction systems in brain tissue obtained from the other Projects. A unique aspect of the Center is the close interaction with two clinical Centers (at the University of Pennsylvania and the University of Virginia) to provide translational capabilities. The Administrative Core will coordinate Center activities, conduct a pilot studies program, and provide for annual meetings with clinical Centers. The Tissue Core will provide uniform sets of brain tissue samples to the projects for individual studies. These projects utilize several animal models and technologies uniquely developed by the Center investigators over the previous years of its funding history.

The Administration Core A represents the central administrative component of the Center for the Neurobiology of Addiction Treatment. Overall supervision of the Center is conducted by Dr. Steven Childers, the director and principal investigator. This supervision is also maintained by the Executive Committee, consisting of the principal Investigators of the various Cores and Projects. Administration of the Center is accomplished by our administrative assistant, Ms. Lucy Fasano. In addition to providing the overall central administrative duties for the Center, Core A is also responsible for maintaining non scientific responsibilities. For example, it provides funds for the annual meeting of the External Advisory Board. The Core provides a clearinghouse of information for the lay community about issues regarding the neuroscience of drug abuse, and provides travel funds for our investigators to attend outreach activities. Core A also coordinates the various training and seminar activities for the Center. In this regard, Core A functions in a crucial role to supplement the science of the other projects with the service and educational components that are vital to the Center's overall purpose. Finally, under the supervision of Dr. Sara Jones, Core A provides funding and administrative support for the Center's Pilot Studies Program.

Center for the Neurobiology of Addiction Treatment Administration Core Summary Dr. Steven Childers, Center Director The Administrative Core is the central administrative component of the Center for the Neurobiology of Addiction Treatment. Overall supervision of the Center is conducted by the Director, Dr. Steven Childers. This supervision is also maintained by the Executive Committee, which consist of the Project and Core Directors, as well as the co-investigators in the Center. Administration of the Center is accomplished by our administrative assistant, Ms. Pam Pitts, who performs financial duties, schedules meetings and supervises outreach activities. One of the primary functions of the Administrative Core is to coordinate all research activities within the Center, maintaining regular meetings of the Executive Committee and the individual Projects. The Core schedules and coordinates tele- and video conferences with our external collaborators, especially our clinical partners. The Core supports and coordinates the annual meeting of our External Advisory Board. The Core also coordinates the Center's data-sharing capability, and maintains the Center's web site. Finally, the Core provides critical resources for training and outreach activities that are vital to the Center's overall purpose.