William L. Dewey - US grants

The overall objective of the research described in this proposal is to elucidate the mechanism of action of morphine and other opiates. The specific aims of the work proposed in this application are: one, to continue to test our hypothesis that the release of endogenous opiates is an important step in the antinociceptive action of morphine. The data which we have generated thus far indicates that there is an increase in the quantity of known endogenous opiates including met- and leu-enkephalin, Beta-endorphin and dynorphin in dog CSF following an acute injection of an antinociceptive dose (10 mg/kg, s.c.) of morphine. We have also presented evidence that this dose of morphine causes an increase in yet to be identified peptides in CSF which also have opioid activity. Our data also suggests that there is at least one nonpeptide with opioid activity in CSF after morphine. This opioid activity is not due to the presence of morphine in the CSF. Secondly, we propose to study the antinociceptive activity of calcitonin and other endogenous peptides not classified as opiates. Another aspect of these studies will be an investigation of the effects of calcitonin and other endogenous peptides on morphine's actions. Our third specific aim is to elucidate the role of endogenous opioid peptides in sudden infant death syndrome. Preliminary results show that the level of immunoreactivity to Beta-endorphin antibody is significantly higher in babies whose siblings have died of sudden infant death syndrome than in infants of the same age who have had spinal taps for other reasons. The basic information generated from these projects will expand our understanding of pain and its alleviation by opiates, the role of endogenous opiates in normal and abnormal breathing, and the role of peptides not considered opioid as neuromodulators in the actions of endogenous and exogenous opiates.

This proposal seeks to procure a reliable and sensitive gas chromatograph-mass spectrometer to be used by a vast number of scientists involved in biomedical research which is supported by the National Institutes of Health. There are other faculty members at MCV/VCU who desperately need a GC/MS to carry out the specific aims of their research which has been funded by other peer review groups of the federal government. Scientists at this University currently are traveling to other cities or sending their samples to other cities to get them analyzed. This is a very expensive and time wasteful way to do research. The machine that was used for these purposes in recent years has an unreliable data system for which we cannot obtain a service contract or replace some of the outdated parts. A new GC/MS would be a stimulus for more research by these productive investigators. The research carried out by these investigators is very broad, including chemical synthesis, molecular, cellular, in vivo animal and clinical research. A number of these investigators are involved with identifying and quantitating endogenous and exogenous substances. The high level of sensitivity provided by GC/MS make this technology essential for this area of research. The proposed instrument has been chosen after careful consideration of all other available and it will best fill the requirements of the researchers involved with this type of methodology.

? DESCRIPTION (provided by applicant): We are requesting continued funding of this productive training program to prepare pre-doctoral and postdoctoral fellows to become independent scholars in studying the biomedical aspects of drugs of abuse. The training record of this program is outstanding, due to the quality of the trainees and preceptors and its organizational structure. Competition for support from this program has been strong and all slots have been full. Trainees are expected to be supported by this program for no more than 3 years. Since 2010, 13 pre-doctorals and 6 postdoctorals have earned individual NIH fellowships. A large number of individuals who received training from these preceptors have progressed to become leaders in the drug abuse field and a very impressive number have earned funding from NIDA and other sources for their substance abuse-related research. Since the submission of our last competitive application we have recruited three new faculty from other institutions and two faculty members from other departments in this university. The recruitment of more clinical faculty is seen as significant. Thus we are requesting support for 10 predoctoral trainees an increase of one, and we will maintain our postdoctoral trainees at 6. Our preceptors provide the trainees with cutting edge expertise in molecular, cellular, behavioral, clinical and translational research. The major emphasis of this program continues to be concentrated on the pharmacology and mechanism of action of abused drugs and mechanisms of addiction. We continue to have a broad scope in our training program and expect that each preceptor will bring his or her specific expertise to the benefit of all trainees. This is accomplished through the wide variety of courses, the high level of collaboration among the mentors, the faculty serving on thesis committees and our excellent seminar series. We also have two new core facilities, one devoted to the development of genetically altered animals and the other to a sophisticated bioanalytical technology. The program has enjoyed and will continue to benefit from strong administrative support from the Department of Pharmacology and Toxicology and the administration of the school and university. The predoctoral training program is rigorous and is characterized by two years of required course work and participation in excellent research projects. Postdoctoral fellows will concentrate on research throughout their training. The major emphasis of all training supported by this program is on research. The faculty has earned the research support necessary for the trainees through the significant number of grants they have generated. We will provide guidance on research, teaching and the professionalism for a career in biomedical science and all aspects of becoming a productive and independent researcher. This will continue to be the hallmark of this program as we continue to provide the atmosphere for the training of leading scholars in the drug abuse field for the future.

nicotine; meeting /conference /symposium; drug addiction; alcoholism /alcohol abuse; publications; behavioral medicine; drug abuse therapy; mental health; travel;

The purpose of this grant is to bring a variety of scientific disciplines to bear on a number of specific drug abuse problems. We propose to carry out major projects in the areas of chemical receptor probes, narcotic agonists and antagonists, the active principles of marihuana and methododlogy development. In the area of chemical receptor probes, we plan to synthesize specific compounds for studies of the postulated "cough", PCP, sigma agonist, nicotine, and THC receptors. In the narcotic areas, we plan to extend our studies toward the elucidation of the "cough" and sigma agonist receptors. We will continue to pharmacologically characterize opiate agonists and antagonists from our synthetic and drug abuse programs. Our studies with the cannabinoids will concentrate on assessing the dependence potential of Delta9-THC, the active principle of marihuana. We also plan to detemrine whether there are specific anatomical sites in the brain responsible for the typical behavioral effects of the cannabinoids. We plan to develop methods for better evaluating the rreinforcing efficacy of drugs of abuse. That is, why do some drugs act as more powerful reinforcers of behavior than others and what can we do to alter this? We will use cocaine as the first drug in these studies.

centrifugation; biomedical equipment resource;

The Committee on Problems of Drug Dependence will hold its 50th Scientific Meeting in North Falmouth, MA. June 26 through July 1, 1988. There will be three days of scientific sessions which will be made up of volunteer oral presentations and poster sessions as well as a number of outstanding timely symposia. The proposed symposium and the respective organizers are as follows: Molecular Mechanisms of Abused Substances - Drs. Eric Simon and Leo Hollister; Mechanisms of Drug Tolerance and Dependence - Drs. E. Leong Way and william Dewey; Long-Acting Narcotic Agonists and Antagonists - Drs. Richard Hanks and Louis Harris; Treatment of Chemical Dependence - Drs. Herbert Kleber, Mary Jeanne Kreek and Charles O'Brien; Current Status of Intravenous Drug Abuse and AIDS - Drs. Beny Primm and Donald DesJarlais; Drugs of Abuse and the Immune System - Drs. Martin Adler and Arthur Falek; Drug Abuse Prevention - Drs. Raymond Lorion and James Anthony. Plenary and award lectures as well as satellite meetings and speciality workshops will complete the program. The proceedings will be published. These proceedings continue to be a valuable compendium of the important work in all aspects of drug abuse research.

The overall objective of this project continues to be the elucidation of the mechanism of action of morphine and other opiate analgesics. A secondary objective is to determine the role of endogenous opioid peptides in various respiratory diseases of children and infants. We have found that children who have apnea as a part of their symptomatology have an increased level of endogenous opioid peptide in their cerebrospinal fluid. We now propose to expand our preliminary results which show that naltrexone, an opiate antagonist, is efficacious in treatment of the disease in these children. We further will explore whether other endogenous opioid peptides, such as met-enkephalin, leu-enkephalin, and dynorphin, are also elevated and a causative factor in the disease of these patients. We will continue to develop an animal model for these diseases which will be essential to evaluate the etiology of the disease and mechanism of action of naltrexone. The second portion of this proposal has to do with our demonstration that the intrathecal injection of mice with morphine produces a pronounced hypoglycemia. This was a surprising observation, since morphine administered by any other route of administration produces hyperglycemia. We will continue to investigate the mechanisms of disappearance of glucose from the blood following the intrathecal injection of opiates. Thirdly, we propose to continue our investigation into the role of intracellular calcium in the mechanism of action of morphine. It has been shown by others and ourselves that morphine alters calcium homeostasis in both the drug-naive animal as well as in the opiate-tolerant animal. These investigations will continue. The work described in this proposal has both theoretical and practical applications. The calcium and glucose experiments will add greatly to our knowledge of the effects of morphine in living organisms, while our studies with the endogenous opioid peptides will be beneficial to the treatment of children with various diseases characterized at least in part by apnea.

The overall hypothesis of this proposal is that neuronal Ca++ homeostasis plays a major role in opioid analgesia and tolerance by affecting signal transduction systems and K+ channel regulation, which ultimately translates into longterm change in endogenous opioid and neurokinin peptide release. The aims of this proposal are to: (1) understand how opioid analgesia and tolerance, and (3) elucidate the role of endogenous opioids and neurokinins in opioid analgesia and tolerance. Regarding Ca++- sensitive systems (1), manipulations intended to increase cytosolic Ca++ block acute opioid analgesia, while Ca++ channel antagonists potentiate analgesia. Alternatively, a disruption in Ca++ homeostasis accompanies the development of morphine tolerance, as evidenced by increases in synaptosomal 45 Ca++ uptake and higher free intracellular Ca++ levels in brain and spinal cored synaptosomes. We propose to test the hypothesis that opioid analgesia and tolerance are modulated by the relative activity of calmodulin and the subsequent activation of calmodulin-sensitive adenylate cyclase (and indirectly PKA activity), and/or multifunctional calmodulin-dependent protein kinase II (CaM kinase II). Another potential contributor to analgesia and tolerance is related to the observation that stimulation- induced increases in cytosolic Ca++ activates phospholipase C (PLC) independent of receptor G-protein coupling. We will test the hypothesis that the phosphatidylinositol system plays a role in opioid analgesia, and that opioid tolerance-induced disruptions in Ca++ homeostasis are related to PLC and the subsequent production of IP3 and activation of protein kinase C (PKC). Regarding K+ channel regulation in analgesia and tolerance (2), we have clearly demonstrated that acute morphine analgesia is antagonized by blockers of potassium channels such as glyburide and apamin. Consistent with this are in vitro observations that K+ channel antagonists prevent mu opioid-stimulated k+ efflux and neuronal membrane hyperpolarization. We will test the hypothesis that chronic administration of mu opioids decreases potassium efflux from neurons as compensatory mechanism in the process of the development of tolerance. Regarding neurokinins and endogenous opioids (3), we will evaluate their roles in analgesia and in the development and regulation of opioid tolerance. We hypothesize that besides playing a modulatory role on pain pathways stimulated by excitatory amino acid neurotransmitters, neurokinins mediate endogenous opioid release. We and others have demonstrated that neurokinin agonists produce a naloxone-sensitive analgesia which we propose results from endogenous opioid release. We will test the hypothesis that opioid tolerance involves changes in spinal neurokinin levels or release, and the neurokinins participate in opioid analgesia by releasing endogenous opioids and altering Ca++ homeostasis. These studies are intended to clarify the role of neurokinins and endogenous opioids in tolerance, since their participation in tolerance is frequently obscured.

Tolerance to morphine involves numerous adaptations at multiple levels of the signal transduction pathway. Studies suggest that changes likely involve desensitization of multiple opioid receptors, decreased G-protein coupling, decreased GTPase activity, increased adenylyl cyclase activity, increased cAMP-dependent kinase activity (PKA) increased protein kinase C activities (PKC), increased Ca2+ conductance, decreased potassium conductance and induction of immediate early genes. Adaptational changes lead to elevated intercellular free calcium levels, which could in turn contribute to the decreased efficacy of morphine in inhibiting neuronal firing and neurotransmitter release. Recent studies in our laboratory have implicated protein kinases, especially PKA and PKC, in the regulation of tolerance. Morphine tolerance can be reversed by PKC and PKA inhibitors within 30 min, when central administered together with morphine. The underlying mechanism that triggers the change in morphine induced antinociception as determined in the animal's response to pain, remains elusive. The rapid onset in the effect indicates that constant Phosphorylation of proteins by kinases is essential to maintain tolerance. PKA and PKC have numerous potential target proteins, which are involved in mu opioid receptor mediated antinociception, and thus, the inhibition of PKA or PKC could interfere at numerous sites in the signal transduction cascade. The fact that two kinase controlled by different effector enzymes independently regulate tolerance suggests that a common step within the signal transduction cascade is altered by both kinases. We propose to induce morphine- tolerance and its reversal in vivo, and use membrane preparations from tolerant and reversed-tolerant animals to characterize the effects of treatment at the receptor, G-protein and effector level, by a combination of pharmacological, functional, biochemical and immunochemical methods. These studies will provide a better understanding, which steps in the signal transduction cascade are crucial for the development and maintenance of tolerance.

DESCRIPTION (provided by applicant): Acutely administered morphine produces antinociception via mu opioid receptor (MOR)-mediated intracellular changes that include a decrease in calcium concentration, and a decrease in the activity of adenylyl cyclase and protein kinases. Chronic administration of morphine results in tolerance to its antinociceptive effects and a reversal in the direction of these intracellular events. Our recent studies have implicated protein kinases, especially cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), as well as other steps in the phosphatidylinositol cascade in morphine tolerance. We have reported that inhibition of either PKA or PKC, blockade of phospholipase C or blockade of 1P3 receptors all cause a reversal of morphine tolerance. In other studies we have elucidated the involvement of ATP-gated potassium channels in the actions of acute and chronic morphine. Again we found diametrically opposite effects in the acute versus chronic treatment regimens. The overall goal of the proposed studies is to utilize pharmacological, biochemical and anatomical approaches to determine the critical cellular events underlying the tolerance that develops to morphine-induced antinociception. We have proposed a comprehensive model that includes the steps in the MOR-mediated signal transduction cascade that we hypothesize are involved in tolerance to morphine-induced antinociception. The proposed experiments will test our hypotheses that constant phosphorylation of proteins is required for the maintenance of morphine tolerance and that inhibition of protein-kinase-mediated phosphorylation or upstream steps in their signaling cascades reverses morphine tolerance. We propose to elucidate the mechanisms that lead to the reversal of analgesic tolerance by examining mu opioid receptor levels and phosphorylation state, G-protein activation, adenylyl cyclase activity, and the phosphorylation state of L- and N-type calcium channels.

[unreadable] DESCRIPTION (provided by applicant): Evidence exists in the literature and from our laboratories to implicate PKC in the mechanism of morphine tolerance. It is clear that its role varies with different levels and durations of morphine tolerance. Its role in tolerance to other opioids has not been studied in detail. The importance of PKC in relation to other neuronal constituents has not been well defined. The overall goal of the work proposed in this application is to carry out a collaborative, comprehensive investigation involving electrophysiological techniques at the cellular level (University of Bristol) with whole animal studies (VCU) to elucidate not only if but also how PKC plays a pivotal role in morphine and other opioid tolerance. Another important goal of the research is to elucidate the relative importance of PKC to other cellular proteins such as PKA, arrestins, GRK etc in opioid tolerance. We propose to study the specific isoforms of PKC and to use knockout mice and siRNA techniques in both laboratories to elucidate the mechanisms involved. Close and continual collaboration between the two groups will provide a mechanism for daily input (email and data transfer) into the design of coordinated experiments in each laboratory. This immediate interchange of result will directly affect the design of the next experiment in the other laboratory working at a different level of integration. The specific aims of the project are to test the hypotheses that (1) PKC plays the predominant role in acute moderate morphine tolerance, and that PKA along with PKC are involved in chronic higher levels of morphine tolerance,(2) that opioids with a higher efficacy at mu opioid receptors produce a non-PKC dependent desensitization at MORs and tolerance to these agents in the whole animal is less easily reversed by PKC inhibitors, (3) GRKs, phosphatases and Gq-coupled receptors play a role in some opioid tolerances and (4) that presynaptic sites contribute to both MOR desensitization and opioid tolerance in mice. The relevance of this research to public health is appreciated when one considers that tolerance to opioids limits their use in treating patients such as those with cancer who have chronic pain. We need to understand the mechanisms of tolerance if we are going to alleviate this problem. Also it is essential that we elucidate the mechanisms of tolerance and dependence if we are going to solve the enormous health problems caused by drug abuse. [unreadable] [unreadable] [unreadable]

For over five decades, a large group of productive investigators at VA Commonwealth University (VCU) has made significant contributions to our understanding of drugs of abuse. The interests of these scientists encompass synthesis, pharmacokinetic, neurochemical, molecular, behavioral and pharmacological characterization of most classes of abused drugs. While productive collaborations have always existed among these investigators, the drug abuse research effort at VCU has been enhanced by the creation of this NIDA Center. The primary objective of the Center is to foster multifaceted basic science research. The Center also will continue to provide a mechanism for uniting scientists from many other disciplines in order to share information and new research ideas regarding abused drugs. In this regard, the Center serves a coordinating role for all drug abuse grants at VCU, including numerous NIDA R01 grants, contracts, a training grant, several individual training awards and a program project. Another major contribution continues to be the training and development of young scientists in drug abuse research. The role of the Core is to provide program management and resources, and to facilitate interaction and cooperation through its administration, drug supply, shared instrumentation, and mouse knock-out facility sub-cores. In addition, a Small Grants Program provides a mechanism to attract established scientists in other fields to drug abuse research and to allow for pilot investigations in response to new drug abuse problems by all junior and senior scientists. The Center serves as a national resource through extensive collaborations and shared information with scientists at the national and international level. In addition, the Center maintains an inventory of cannabinoid analogs that we distribute to scientists at VCU and to NIDA-sponsored researchers throughout the US and the world. Of course, basic research will continue to be the primary focus of the Center. The five proposed projects address the mechanisms through which endocannabinoids produce a blockade of morphine tolerance, decrease seizure threshold following chronic cannabinoid administration, and affect the reward and withdrawal of chronic nicotine. These efforts are complemented by studies to understand the plasticity of cannabinoid receptors and characterization of cannabinoid properties of orphan G-protein coupled receptors. Coordination among the projects is directed toward the nature of ligand receptor interactions, roles of receptor-associated proteins, receptor phosphorylation states, characterization of new receptor subtypes, and signal transduction mechanisms. We are also investigating the interactions between the endocannabinoid system and opioids and nicotine. The roles of the nicotinic receptor subtypes in the development of reward and dependence will also be addressed. This center has much significance on human health since additional knowledge of this endogenous system will improve our ability to treat disease.

Morphine and other opioids remain the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. The overall goal of this study is to elucidate the mechanisms of morphine and other opioid-induced tolerance in the ileum and brain but not in the colon which leads to constipation and limits the chronic use of these excellent pain relievers in man. This proposal represents a collaboration of two senior laboratories, one with expertise in gastrointestinal physiology and the other with extensive experience in studying the acute and chronic effects of morphine and other opioids, utilizing molecular, cellular and whole animal approaches. The main hypothesis to be tested is that differences in the cellular signaling properties between the colon and ileum result in morphine-induced tolerance in the ileum but not the colon. We further propose that the ratio of tolerance to various opioids is the same across these tissues. Our preliminary data demonstrate that as marked tolerance develops to the antinociceptive effects of morphine following chronic administration in mice, tolerance does not develop to its constipating effects. The major objective of the work proposed in year 1 is to elucidate whether this difference in tolerance in different tissues observed for morphine is the same for other opioids both in vivo and in vitro. In the second year we propose to further evaluate our interesting finding that nicotinamide, an endogenous CD-38 inhibitor, induces tolerance to morphine in the colon, a phenomenon not previously reported. We will test the hypothesis that the CD-38 pathway prevents tolerance development in the colon and that its blockade by either chemical means or genetically in knock out animals allows tolerance to occur. We propose to investigate whether CD38 in other tissues such as the ileum or brain causes a regulatory effect on the development of tolerance in these tissues as well. Further, we will investigate whether additional steps in the signal transduction process such as the phosphorylation of mu opioid receptors, the activity of protein kinase C and A, GRK alter opioid tolerance development similarly or differently in various tissues. Our preliminary data demonstrate that inhibition of the CD38 pathway by either nicotinamide or 8-bromo cyclic ADPribose results in the induction of tolerance to morphine in the colon. We will test the effects of nictoinamide, ryanodine and cADPribose on tolerance development and on dependence to morphine in the isolated colonic muscle strips and determine if inhibition of the CD38 pathways results in changes in mu receptor phosphorylation in enteric neurons. The long-term major public health implication of this research is the use of this information to develop medications to treat chronic pain that are devoid of constipation. These studies will also increase our understanding of the potential mechanisms in the development of tolerance and ultimately physical dependence to opioids in the brain.

DESCRIPTION (provided by applicant): Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long-term use of this excellent pain reliever in man is limited by side-effects that include analgesic tolerance and opioid-induced bowel dysfunction, with constipation being the most common and debilitating symptom. The long-term goals of this study are to elucidate the mechanisms that lead to tolerance to many of the effects of opioids, including slowing of gastrointestinal transit but not constipation. The main hypothesis to be tested is that differences in the cellular signaling properties of morphine determine the development of tolerance in the ileum but not the colon. Preliminary data suggest that morphine tolerance in the ileum is associated with an uncoupling of the ? opioid receptor from its downstream signaling proteins. Unlike the ileum, the colon which is the major site for constipation does not develop tolerance to repeated administration of morphine. The major objective of specific aim 1 is to test the hypothesis that down-regulation of ? arrestin2 is associated with morphine tolerance in the ileum. The specific goals are to characterize the concentration and temporal relationship for ? arrestin 2 downregulation and tolerance development. Functional and biochemical studies will be utilized to correlate the effect of chronic morphine in-vitro and in-vivo utilizing ?-arrestin2 knock-out mice. Specific Aim 2 will test the hypothesis that ? arrestin2 acts as a scaffolding protein to regulated downstream signaling including MAP kinase, Src kinase, Akt and protein kinase C. Preliminary findings suggest that unlike morphine induced antinociceptive tolerance, in the ileum downregulation of phospho-ERK correlates with tolerance development suggesting fundamental differences in the mechanism for opioid tolerance in the gastrointestinal tract from CNS. This aim will also examine if downregulation of ? arrestin 2 is mediated via altered ubiquitination. Specific Aim 3 will explore the effect of long-term morphine on isolated enteric neurons from the adult mouse myenteric plexus. In this aim, single enteric neurons from the colon and ileum will be characterized and tested to determine morphine- induced changes in electrical excitability and effects on sodium, calcium and potassium channels in wild-type and ? arrestin2 knock-out mice. The information obtained from these studies will increase our understanding of the mechanisms of opioid tolerance and ultimately physical dependence in the gastrointestinal tract and in the brain.

DESCRIPTION (provided by applicant): There is considerable evidence in the literature that deaths due to the simultaneous abuse of ethanol with opioids such as heroin or prescription opioids leads to lower blood levels of the opioid upon autopsy than is observed following death due to the opioid alone. We have carried out an extensive study which has shown that either ethanol or diazepam at doses that do not alter the acute effects of morphine reversed the antinociceptive tolerance produced by chronic morphine. The mechanism of the reversal produced by the two drugs differed in that inhibitors of either GABAA or GABAB receptors alone did not reverse the effect of alcohol but blocking both type GABA receptors completely reversed the effect of ethanol on morphine tolerance. Conversely the inhibition of morphine tolerance produced by diazepam was completely reversed by a GABAA inhibitor alone but not by the GABAB inhibitor. The overall objective of the work described in this proposal is to elucidate the cellular mechanisms by which opioid tolerance is reversed by the concomitant administration of ethanol and other agents affecting the central nervous system. Using genetically altered mice and a number of molecular techniques we will elucidate further the role of GABA receptors and the signaling mechanisms involved in these effects. In the second specific aim we propose to elucidate whether ethanol and diazepam reverses the tolerance to opioids such as buprenorphine and methadone which are used in maintenance therapy and oxycodone and other prescription opioids with a history of abuse. We and others have reported differences in the mechanism of tolerance development for various opioids. For instance, protein kinase A and protein kinase C inhibitors but not GIRK inhibitors reversed tolerance to morphine and other moderately efficacious opioids but not to the high efficacy DAMGO. GIRK inhibitors revered the tolerance to DAMGO but not the other opioids. These differences in the mechanism of tolerance development to various opioids cause us to investigate whether ethanol and/or diazepam will reverse the tolerance to opioids of various efficacies. Obviously, we will elucidate the differing mechanisms if the opioids are affected differently by ethanol or diazepam. In the third specific aim we will investigate whether ethanol and diazepam also reverse tolerance to a peripheral effect of opioids. We and others have found certain differences between the mechanisms of opioid tolerance in the brain and the gastrointestinal tract and now we propose to determine whether ethanol and/or diazepam reverses opioid tolerance in the gastrointestinal tract. The information gained from these experiments will provide important information as we and others continue to elucidate the mechanism of opioid tolerance.

The objectives of the pilot project core are to provide funding for innovative new research to test the hypotheses of investigators to generate preliminary data for future external grant submissions. Young investigators, those who have been funded by external sources for their work in the drug abuse field but have a new hypothesis that needs testing for its feasibility before seeking external funding, investigators who have been successful working in another area of biomedical research and have a hypothesis that utilizes that expertise to test a hypothesis in the drug abuse field. Collaborative projects among scholars from more than one of these groups will be given priority. Scholars from throughout the university will be encouraged to participate in this program and the Department of Pharmacology and Toxicology will provide matching funds for all pilot projects funded. The scholars funded by this mechanism will have an advantage in seeking support from the School of Medicine Research Funding, the campus-wide A.D. Williams Research Program and the Presidents Research Initiative Program. All levels of the university administration will continue to be committed to support drug abuse researchers through this center of excellence and these other mechanisms It is impressive that scholars supported by the pilot project programs in the past have had at least a three times higher success rate with their external applications than the national average.

DESCRIPTION (provided by applicant): The overall aim of this Center is to provide molecular, genetic, chemical and analytical knowledge and expertise to create new and enhance previously funded drug abuse research at this and neighboring universities. These Cores will provide up-to-date and innovative expertise that is somewhat oblique to the methodologies used in previously funded research. This will result in new collaborative research and enhance previously funded research in ways that could not have been contemplated just a few years ago. Cores in synthetic and analytical chemistry, genetics, genetically engineered altered mice/viral vectors, and receptor function will provide the opportunity for funded researchers to broaden the scope of their work to transform knowledge in very creative ways. The inclusion of chemistry, genetics and analytical Cores will fill a major void in our many research projects. The ability to continue to provide genetically altered mice with appropriate genotyping, etc., and the inclusion of expertise in viral vector technology are seen as major assets of this Center. Being able to have a collaborator elucidate receptor function changes for a substance found to alter behavior or craving for a drug provides the atmosphere for new and creative research. This type of collaboration among scientists at different institutions is rare and would take many months or years if in fact collaboration could be established at all. In this Center this type of collaboratin will be the norm. We are committed to provide considerable institutional support such as the use of an up-to-date imaging center and a forensic toxicology laboratory established by the university. We will match dollar for dollar the pilot project program and provide administrative support beyond the modest amount requested. The Cores will also be utilized by NlDA-funded investigators at the University of Virginia and George Mason University. Scholars at the relatively new Pharmacy School at Hampton University have expressed interest (letter from the dean) in the utilization of these Cores as they establish their research programs. We will also share all aspects of this Center with scientists throughout the country.

The administrative core will provide the leadership to ensure that this center continually stimulates new, significant and innovative research while simultaneously providing new cutting edge approaches to enhance the goals of the research projects funded to investigators at this and neighboring institutions. The director of this center will have the responsibility for all aspects of this center but will be provided with daily input from the internal advisory committee some of whom have been colleagues for decades, and frequently from the external advisory committee. In addition the individual core leaders will serve in an advisory capacity with the major goal to stimulate new innovative research available due to collaboration among the cores. This core will rely heavily on continual interaction and communication among all scholars and advisors as they have done for years. There has been a long history of collaboration among the drug abuse researchers at this and neighboring institutions and this experience will provide the essence for the leadership of this center. Should it be necessary the internal advisory committee for this center along with the dean of the School of Medicine will designate one of the core directors or another senior investigator in the drug abuse field to become the center director. The administrative support for this center will be provided by the secretarial and financial personnel as requested in this application but they will have the very large and experienced administrative staff of the Department of Pharmacology and Toxicology as needed. Further, the offices of the School of Medicine and The Vice President for Research have and will continue to provide support at their respective levels.