Laura Sim-Selley - US grants

The proposed studies will examine the effects of chronic ethanol on G- protein-coupled receptor function. Neurotransmitter receptors in specific brain regions are thought to be affected by ethanol. The G- protein-coupled receptors constitute a major class of receptors in brain, and changes in G-protein levels have been detected after chronic ethanol administration. However, there is little data regarding the effects of chronic ethanol on receptor-coupled G-protein activity. The proposed studies will determine whether changes in the functional activity of G-protein-coupled receptors in specific brain regions are affected by a chronic ethanol diet. Initial studies will involve an anatomical survey of the activity of 5-HT1A and opioid receptors in brains from rats that receive ethanol and control rats. In addition, the activity of several G-protein-coupled receptors in the cerebellum will be examined. These experiments will use a novel technique that directly measures receptor-mediated G-protein activation in an anatomically and pharmacologically specific manner: in vitro autoradiography of agonist-stimulated [35S]GTPgammaS binding. Regions in which receptor-G-protein function is altered by ethanol administration will then be examined in [35S]GTPgammaS membrane binding assays. Membrane [35S]GTPgammaS binding assays will allow the determination of changes in the potency or maximal effect of agonists after ethanol administration. Saturation analysis of basal and agonist- stimulated [35S]GTPgammaS binding will be used to determine whether changes occur in the catalytic activation of G-proteins by receptors, or in the affinity of the receptor-coupled G-proteins for GTPgammaS, after chronic ethanol administration. These studies will be combined with receptor binding assays using radiolabeled receptor antagonists. Saturation analysis of receptor binding will be performed to measure any changes in receptor number in those systems that are affected by chronic ethanol administration. The purpose of this R03 application is to provide data that will be used to develop an R01 application in the future by allowing the generation of hypotheses regarding the effects of ethanol on G-protein-coupled receptor functional activity.

DESCRIPTION (provided by applicant): Cannabinoid (CB1) receptors in brain mediate the effects of delta 9-tetrahydrocannabinol (THC) and endocannabinoids, primarily by activation of inhibitory G-proteins of the Gi/Go family. However, it is our premise that CB1 receptor- G-protein coupling is not uniform throughout the brain, and CB1 receptors may couple to multiple G-proteins to account for the multiplicity of cannabinoid effects. Indeed, our progress to date demonstrates regional differences in CB1 receptor-mediated G-protein activity in both normal brain and in adaptive responses to chronic THC administration. Moreover, the pattern of tolerance development is not identical for all THC effects, a finding we hypothesize is related to these regional differences in CB1 receptor-G-protein coupling. The proposed studies will investigate the relationship between cannabinoid tolerance and CB1 receptor desensitization and downregulation, and examine whether selective coupling of CB1 receptors to specific G-protein subtypes is correlated with regional differences in CB1 receptor adaptation. We propose examining in a systematic manner the role of CB1 receptor- G-protein coupling in neuroadaptation not only as a means of understanding cannabinoid tolerance but as a way of characterizing the endocannabinoid system. In order to address the role of receptor occupancy in adaptation, the magnitude of THC tolerance will be varied by administering different doses of THC. We will then assess 1) tolerance to cannabinoid-mediated hypoactivity, antinociception, hypothermia and memory impairment in behavioral assays and 2) CB1 receptor downregulation and desensitization using radiolabeled ligand and agonist- stimulated [35S]GTPgammaS autoradiography. We also hypothesize that differences in CB1 receptor-G-protein coupling throughout the brain account for differences in recovery of tolerance to separate THC-mediated behavioral effects. Therefore, the temporal relationship between recovery of tolerance and CB1 receptor function will be evaluated by treating mice with THC, then evaluating tolerance and downregulation/desensitization at different times after cessation of treatment. We will also conduct experiments to determine whether CB1 receptor coupling to different G-protein subtypes is responsible for variations in cannabinoid actions in different brain regions. We will examine co-localization of CB1 receptors and specific G-beta and G-gamma subtypes using immunocytochemistry to determine whether there is selective co-localization of CB1 receptors and specific subunits in different regions. We will then examine whether chronic THC administration selectively alters CB1 receptor coupling to specific G-alpha subtypes using agonist- stimulated [35S]GTPgammaS binding with subsequent immunprecipitation of activated G-alpha subtypes. These studies will contribute to elucidation of the mechanisms of action of CB1 receptors in brain, as well as determine the effects of chronic cannabinoid administration on cellular function during tolerance.

DESCRIPTION (provided by applicant): Pain is a multi-faceted, complex disease that affects all humans. Unfortunately, progress in pain management has been met with limited success. However, considerations of the multiple components of pain have suggested that targeting non-conventional sites could strongly impact the pain management field. The endocannabinoid (eCB) system is one of several lipid signaling systems in the brain and in the body. Verified components of this system include two G-protein coupled receptors, their signaling pathways, two predominant endogenous ligands [anandamide (AEA) and 2-arachidonyl glycerol (2-AG)], and their synthetic and metabolic pathways. The system plays an important modulatory role in many crucial CNS processes (e.g., brain reward, appetite regulation, cognition). Consequently, it is not surprising that this system has been implicated in the pathophysiology of a variety of health problems related to these processes, including pain management. This application is largely based on the idea that a clinically significant component of pain is behavioral depression (i.e., pain-depressed behaviors). In humans, this is indicated by absences from work or school, lack of interest in customary activities, overall decreases in motor activity, and is most often associated with clinical depression. In animals clinical approximation of pain is through decreases in locomotion or grooming and interest in feeding or social interaction. Given these, a promising new strategy for comprehensive treatment of pain is an adjunct focus on pain-depressed behaviors and depressed mood. With this application we plan to evaluate eCB modulation of pain-depressed behaviors using intracranial self-stimulation (ICSS) and drug discrimination (DD) in mice. ICSS has been widely used to study modulation of motivated behavior (i.e. reward) and affect by drugs whereas DD is primarily used to model the subjective/intoxicating effects of drugs. We propose utilizing these well-established operant procedures to evaluate the eCB's effects on pain-induced behavioral depression, affect and intoxication. To complement these behavioral measures, we will determine mechanistic characteristics of affective cannabinoid analgesia versus reward in selected brain regions such as the nucleus accumbens, a brain area implicated in reward and affective pain, through the use of well- established neurochemical analyses such as mass spectrometry and [35S]GTPgS G-protein binding studies. Given the clear need to explore novel therapeutic targets, improve upon existing preclinical pain assays, and incorporate the affective component of pain, we propose that studying the eCB system's modulation of pain-depressed behavior will meet these needs. We feel these studies have significant public health implications and offer a large degree of innovation while relying upon well-established behavioral and neurochemical measures. In summary, considering the paramount public health concern regarding effective pain management this application promises to establish whether the eCB system is a viable and attractive therapeutic means to effectively reduce the great societal burdens associated with pain management. PUBLIC HEALTH RELEVANCE: Pain is a public health concern of utmost importance and is typicaly accompanied by behavioral depression that results in absences from work/school, lack of interest in customary activities, overall decreases in activity, and is most often associated with depresion. Given the clear need to explore novel therapeutic targets, improve upon existing preclinical pain assays, and incorporate the affective component of pain, we propose a thorough evaluation of the endogenous cannabinoid system's ability to ameliorate pain-depressed behavior, while taking into account critical factors relevant to this system such as modulation of reward and intoxication.

? 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.