Aron H. Lichtman, PhD - US grants

Though the effects of naturally occurring and synthetic cannabinoids have been well described, their mechanisms of action are just beginning to become known. Recent breakthrough demonstrating differences between the distribution of cannabinoid receptors and the receptor mRNA in the CNS strongly suggest the existence of cannabinoid neuronal pathways. The objectives of the research proposed in this project are to: 1) identify specific brain region which mediate the behavioral effects of the cannabinoids, and 2) characterize cannabinoid neuronal pathways. These proposed studies will focus on the hippocampus and basal ganglia because, in addition containing high concentrations of cannabinoid receptors and cannabinoid receptor mRNA, they mediate many of the behaviors associated with cannabinoid activity. The behavioral indices which will be assessed are the nociception, catalepsy, short-term memory, spontaneous activity, hypothermia, and rotational behavior. Using a pharmacological approach, which will include dose-response and SAR studies, the effects of cannabinoids in specific brain sites will be examined. In a separate series of studies, the neuronal pathways will be characterized by selective lesions of specific brain structures containing high concentrations of cannabinoid receptors or cannabinoid receptors mRNA. The methods of autoradiography and in situ hybridization histochemistry will then be used to assess changes in receptor number and expression of cannabinoid receptor mRNA, respectively. The results of this research will be an important first step in the elucidation of the neural substances of cannabinoid activity.

This mentorship program is designed to increase the pool of exceptional individuals interested in pursuing a career in the biomedical sciences. Disadvantaged high school students are recruited from all the schools in the Richmond, Virginia metropolitan area. They are selected on the basis of grates, letters of recommendation, and expressed personal interest as stated in essays. K-12 teachers and teachers-in training are recruited from the same geographical area and are selected on the basis of expressed interest, letters of recommendation, and personal interviews. All trainees participate in a ten week summer research program under the supervision of a diverse multi-disciplinary group of biomedical scientists. This experience includes both oral and written research presentations. The high school students are required to keep a daily journal of their experiences in the laboratory. In addition, all participants attend weekly seminars focusing on various topics of research or careers in science. Finally, the students are provided the opportunity to broaden their knowledge of biomedical research throughout the academic school year by attending research seminars, participating in Saturday morning science programs, attending the "minimed school", and participating in regional and state science competitions. Each participant is assigned to a mentor who will serve as a guide and advisor. The mentors are available to advise the students after high school and college graduation. Each of the K-12 teachers and teachers-in training will meet weekly with 3 or 4 of the students during which time they discuss each other's research. In addition, the teachers are expected to share their experience and knowledge of biomedical research to their classrooms. They are provided resources and advise in preparing lesson plans and laboratory experiments for use in their schools. We follow the students through high school, college and hopefully graduate or professional school.

DESCRIPTION (provided by applicant): The discovery of an endocannabinoid system in the brain consisting of cannabinoid CB1 receptors and endocannabinoids (e.g., anandamide and 2-AG) has generated a great deal of interest in understanding the physiological functions of this system. Based on converging in vivo and in vitro evidence this system has been proposed to play an active role in processes that underlie the degradation of memory over time (i.e., forgetting) as well as the suppression of learned behaviors that are no longer reinforced (i.e., extinction). Under normal circumstances, these processes are further hypothesized to facilitate the encoding of new information. The studies proposed in this application will examine the function of this system by blocking endocannabinoid signaling either permanently (i.e., CB1 (-/-) mice) or acutely through the use of the CB1 receptor antagonist SR 141716. Although these approaches can indirectly implicate the involvement of endocannabinoids, the availability of mice in which fatty acid amide hydrolase (FAAH), the primary enzyme responsible for anandamide metabolism, has been genetically deleted (i.e., FAAH (-/-) mice) along with a selective FAAH inhibitor will enable us to determine whether this endocannabinoid plays a role in memory. Additionally, we will investigate the neural substrates and receptor mechanisms of action that underlie endocannabinoid modulation of memory. We will ascertain whether the levels of anandamide and 2-AG are modified by behavioral procedures that are found to be under endocannabinoid tone. Finally experiments will also be conducted to determine whether the mnemonic effects of the cannabinoids are mediated in the hippocampus, a brain region that not only contains CB1 receptors and endocannabinoids, but also is known to play a role in learning and memory. Collectively, these studies will further our understanding of the physiological function of this system as well as bridge the gap between the in vitro and in vivo actions of the endocannabinoid system.

DESCRIPTION (provided by applicant): Marijuana has been the most prevalent illicit drug of abuse for many years; yet, major questions remain regarding the health consequences of long-term use. The pharmacological and toxicological properties of smoked marijuana have not been thoroughly investigated because of the challenges posed by inhalation studies, particularly with a smoked plant material. Instead, the research emphasis has been on the major psychoactive constituent in marijuana, delta-9-tetrahydrocannabinol (THC). The gap in our knowledge has led to speculation that constituents other than THC contribute to marijuana's effects. Equally important is the question of what adverse effects result from long-term use of marijuana for either recreational or medicinal purposes. Our first goal is to determine whether the effects of marijuana are due solely to THC. We will expose mice acutely to marijuana smoke as well as THC vapor to determine whether the effects on motor activity, pain sensitivity, body temperature, and memory are identical. We will examine marijuana samples with different quantities of cannabinoids, as well as spike them with known cannabinoids. Blood and brain levels of THC will be determined by mass spectrometry to establish dosimetry and pharmacokinetic factors. The second goal will be to determine whether chronic exposure of marijuana smoke and THC inhalation in mice produces similar degrees of drug dependence, cognitive impairment, immunosuppression in lung, and tumor proliferation. Mice will be exposed repetitively to marijuana smoke or THC inhalation for either one week, two weeks, and three months. At the end of each exposure period, they will be challenged with the CB1 receptor antagonist SR 141716A to assess the degree of dependence. The Morris water maze will be used to assess alterations in reference and working memory. Finally, immune function and tumor proliferation, as well as the histopathology, will be evaluated in the lungs of animals exposed for varying periods of time. The discovery of the central and peripheral endocannabinoid systems consisting of CB1 and CB2 cannabinoid receptors, signal transduction pathways, endocannabinoids [i.e., anandamide (AEA) and 2-arachidonyl glycerol (2-Ara-GI)], putative AEA membrane transporter (AMT) and AEA's metabolic enzyme fatty acid amidohydrolase (FAAH), provides an opportunity to determine which effects of marijuana smoke are mediated through this system

Brain; Endocannabinoids;

The goal of this project is to address several unresolved questions regarding the role of the endocannabinoid system in pain, reward and drug dependence. There is strong evidence that the two major endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) exert considerable influence in these pathological states, yet it is highly unlikely that they are simply playing redundant roles. Therefore, one of the objectives of this project is to delineate their roles in these pathological conditions. The fact that the synthetic and metabolic pathways for endocannabinoids are not completely understood creates a challenge in systematically manipulating their levels under in vivo conditions. Therefore, a major goal is to develop potent and selective enzyme inhibitors that can be used to manipulate endocannabinoids in vivo. Synthetic and metabolic enzyme inhibitors for AEA and 2-AG will be prepared by Dr. Razdan (Project 2). Those identified by Dr. Cravatt (Project 3) as enzyme selective will be evaluated by us in behavioral battery of tests for cannabinoid activity. The second approach is to establish the phenotypes of mice deficient in endocannabinoid synthetic and metabolic enzymes generated by Dr. Cravatt. At the same time, we are well aware that several classes of lipids structurally related to AEA and 2-AG impact the endocannabinoid system by influencing synthetic and metabolic pathways, acting directly on cannabinoid receptors, or acting at the newly discovered allosteric site on the CB{1} receptor site. To address these questions, we will conduct in vitro and in vivo evaluation of synthetic allosteric ligands for CB{1} receptors [prepared by Drs. Razdan and Mechoulam (Project 4)], and putative endocannabinoids provided by Dr. Mechoulam. To complement Dr. Mechoulam's efforts to identify new endocannabinoids, we will establish lipid profiles in selected brain regions of mice under different experimental conditions in an effort to identify lipids that may be endocannabinoids or relevant lipid mediators. We will use existing as well as these new discoveries to further explore the involvement of the endocannabinoid system in pain, reward and dependence. Emphasis will be placed on inflammatory and neuropathic pain models. We will determine the extent to which cytokines and chemokines are involved in endocannabinoid anti-inflammatory effects. The receptor mechanisms of action and underlying neural substrates will be investigated using genetic and pharmacological approaches. The same comprehensive approach will be employed in drug discrimination and feeding behavior, to establish THC-like profiles of agents that manipulate the endocannabinoid system and to establish phenotypic behavior of genetically modified mice. Collectively, these studies will identify endogenous substances that may act either directly or indirectly on the endogenous cannabinoid system, and elucidate the role that AEA and 2-AG play in pain, reward, and drug dependence.

DESCRIPTION (provided by applicant): The primary objectives of this Program Project are to establish the functional roles of the endocannabinoid system in normal physiological processes and in abnormal or disease states and to determine the extent to which it serves as a mediator in the effects of exogenous cannabinoids. It is our premise that understanding the endogenous cannabinoid system will allow us to address public health issues regarding drugs of abuse as well as co-morbidities that include cognitive disorders, compulsive behavior, pain, motor dysfunction and numerous peripheral disorders. Incredible progress in the past decade has firmly established the existence and homeostatic importance of the endocannabinoid system and its key components (receptor subtypes, endogenous ligands, synthetic and metabolic pathways, signaling pathways, etc.) As with most biological systems, as knowledge accumulates so does the level of complexity. We have assembled a team of chemists (Mechoulam, Razdan and Mahadevan), biochemists and molecular biologists (Cravatt and Di Marzo), and pharmacologists (Martin, Wiley, Lichtman, Pertwee, and Ross) who will address fundamental questions related to distinguishing between the physiological functions of anandamide (AEA) and 2- arachidonoylglycerol (2-AG), investigating whether other endocannabinoids exist, and determining the relationship between the roles of endocannabinoids in neural processes (e.g., pain, reward, neuroprotection) and peripheral processes (e.g., bone formation, inflammation). Our approach involves synthesis of putative endocannabinoids, stable and potent analogs, metabolic and synthetic enzyme inhibitors and CB1 receptor allosteric modulators. Furthermore, we will manipulate enzymes that are putatively responsible for AEA and 2-AG synthesis and degradation as well as establish the resulting lipid profiles using liquid chromatography, ion trap, time-of-flight mass spectrometry to identify new lipid entities associated with the endocannabinoid system. Dr. Cravatt will systematically delete existing as well as proposed synthetic and metabolic enzymes for AEA and 2-AG in mice. These genetically modified animals, along with the synthetic probes and newly discovered endocannabinoids, will be used to further elucidate the function of the endocannabinoid system in neuropathic and inflammatory pain, neurotrauma, reward, and dependence-related events. Ultimately, the knowledge gained from this basic research will yield novel therapeutic targets that can be exploited with the pharmacological agents developed here. PROGRAM CHARACTERISTICS

DESCRIPTION (provided by applicant): The endogenous cannabinoid system is a neuromodulatory system that affects a wide range of physiological processes, including memory and inflammation. Evidence indicates that exogenous and endogenous cannabinoids are neuroprotective and thus there is interest in the therapeutic potential of cannabinoids in Alzheimer's disease. In vitro studies have shown that cannabinoid receptor activation can inhibit or reduce the deposition of beta-amyloid plagues and decrease inflammation, critical features of Alzheimer's disease. Moreover activation of the cannabinoid system has neuroprotective effects. While these findings indicate that cannabinoids may be beneficial in attenuating the neuropathology associated with Alzheimer's disease, very few studies have evaluated if stimulation of the endocannabinoid system can attenuate cognitive deficits and neuropatholgy in in vivo models of Alzheimer's disease. The goal of these studies will be to examine whether elevating endogenous levels of the endocannabinoid anandamide via inhibition of its primary degradative enzyme fatty acid amide hydrolase (FAAH), will have beneficial effects on memory impairments and neuropathological markers in 3xTg mice, an in vivo model of Alzheimer's disease. More specifically, we will determine whether repeated administration of the FAAH inhibitor PF-3845 improves learning and memory and prevents or delays neuropathological processes of Alzheimer's disease. The findings from these proofs of principle studies may lead to the identification of a new therapeutic agent for memory deficits and neuropathology associated with Alzheimer's disease.

PROJECT SUMMARY (See instructions): The objective of this Core is to provide expert advice from the talented team of co-investigators, necessary training, and the infrastructure to utilize molecular biological approaches to create genetically engineered mouse models that will increase the depth and breadth of existing drug abuse research and stimulate new research at Virginia Commonwealth University and throughout the scientific community. Additionally, it will provide a mechanism for scientists both within and outside of the drug abuse field to interact to develop new research projects. Specifically, this Core will provide investigators the necessary training, tools, and expertise to: 1) create novel genetically modified mice, not currently available through other sources; 2) maintain mice in a repository in which available mouse lines will be bred, genotyped, and transferred to the investigator upon weaning; 3) cryogenically preserve mouse lines for use in future studies; 4) develop viral vectors to over-express, knock-down or deliver dominant negative forms of study genes chosen by collaborating investigators; and 5) deliver viral vectors stereotaxically into specific brain regions of interest and verify the extent of the manipulation. Overall this Core will provide new services to multiple investigators, with the goals of providing state-of-the-art molecular biological tools to investigate drugs of abuse, facilitate collaborations, and provide value to drug abuse researchers.

? DESCRIPTION (provided by applicant): Chronic pain diminishes the quality of life for millions of patients, but currently used classes of analgesics possess varied efficacy and are associated with a variety of untoward side effects. Thus, novel targets to treat chronic pain and development of new drugs that have better efficacy and/or fewer side effects than existing pharmacotherapies are greatly needed. A particularly promising target is the sphingosine-1-phosphate (S1P) receptor system, which mediates CNS neuromodulatory functions. FTY720-phosphate, the active metabolite of FTY720 (FTY; fingolimod), approved by the FDA for treatment of relapsing multiple sclerosis, acts as an agonist at four of the five S1P receptors (S1P1, 3, 4, 5). Interestingly, studies have demonstrated that FTY and other S1P receptor (S1PR) agonists produce antinociception in acute thermal rodent pain models and these effects are blocked by central administration of an S1P1-selective antagonist. Moreover, FTY reverses hyperalgesic states in rodent neuropathic pain models. However, it is unclear whether S1P1 or other S1PR subtypes mediate these effects and their site(s) of action. Thus, the overarching hypothesis of this application is that the S1P1 receptor represents a novel and promising target for the treatment of neuropathic pain. Here, we will test whether S1P1 receptors in the CNS mediate anti-hyperalgesic effects in a mouse neuropathic pain model, using a combination of pharmacological and gene targeting approaches. Therefore, the Specific Aims are to: 1) Determine the role of S1P1Rs in alleviation of neuropathic pain by S1PR ligands; 2) Determine the role of FTY-induced S1PR adaptation in FTY-mediated reversal of neuropathic pain; and 3) Determine the role of S1P and S1P1 receptors in spinal glia in CCI-induced neuropathic pain and its reversal by FTY. The studies proposed herein will establish whether FTY and selective S1PR ligands reverse pain-related behavior in the mouse CCI neuropathic pain model, whether S1P1 receptors in the nervous system mediate these actions and the specific cell types involved in the response. In order to be useful in treating chronic pain, the drug must retain its effectiveness during prolonged treatment. Thus, evidence supporting a role of S1P1 in specific cell types to reduce neuropathic pain without tolerance or motor impairment will provide proof of principle that S1P1 receptors are a viable target to treat neuropathic pain and possibly other chronic pain-related disorders.

PROJECT SUMMARY Please see Research Strategy section.

Project Summary ? Mutant Mouse/Viral Vector Core The objective of this Core is to provide expert advice from the talented team of co-investigators, necessary training, and the infrastructure to utilize molecular biological approaches to create genetically engineered mouse models that will increase the depth and breadth of existing drug abuse research and stimulate new research at Virginia Commonwealth University and throughout the scientific community. Additionally, it will provide a mechanism for scientists both within and outside of the drug abuse field to interact to develop new research projects. Specifically, this Core will provide investigators the necessary training, tools, and expertise to: 1) create novel genetically modified mice, not currently available through other sources; 2) maintain mice in a repository in which available mouse lines will be bred, genotyped, and transferred to the investigator upon weaning; 3) cryogenically preserve mouse lines for use in future studies; 4) develop viral- vectors to over-express, knock-down or deliver dominant negative forms of study genes chosen by collaborating investigators; and· 5) deliver viral vectors stereotaxically into specific brain regions of interest and verify the extent of the manipulation. Overall, this Core will provide new services to multiple investigators, with the goals of providing state-of-the-art molecular biological tools to investigate drugs of abuse, facilitate collaborations, and provide value to drug abuse researchers.