John T. Williams - US grants

Synaptic transmission mediated by monoamines is terminated by a reuptake process that moves transmitter from the extracellular space into the cytoplasm. The major biochemical action of cocaine is to inhibit the activity of transporter proteins for noradrenaline (NA), dopamine (DA) and 5-HT. Thus cocaine acts to prolong the presence of transmitter extracellularly. These transporter proteins are concentrated at nerve terminals of monoamine cells, such that these terminals are key sites to investigate the actions of cocaine. Studies of the cell bodies of monoamine containing neurons in vitro suggest the there is continuous release of NA in the locus coeruleus, DA in the ventral tegmental area and 5-HT in the dorsal raphe. The inhibition of reuptake by cocaine increases the extracellular concentration of transmitters which activate autoreceptors that hyperpolarize these monamine cells. In contrast, there is not effect of cocaine on the membrane potential of cells in unstimulated preparations in areas innervated by these monoamine cells. Postsynaptic potentials are often used as a sensitive assay for transmitter release from presynaptic terminals. We will measure the amplitude and duration of synaptic potentials to study the modulation of transmitter release by cocaine. One aim of this proposal is to determine how cocaine modulates the release of monoamines from terminals. Cocaine prolongs monoamine-mediated synaptic potentials and also decreases the amount of monoamine released. The mechanism for the inhibition of monoamine release is not known. Each of the cell body regions previously studied receives projections from other monoamine nuclei. Using intracellular recording from cells in the brain slice preparation. The effects of cocaine will be studied in the ventral tegmental area and dorsal raphe. This study will focus on the interaction between S-HT, dopamine and cocaine in the VTA and the factors that regulate activity in the dorsal raphe. Knowledge of the actions of cocaine at terminals is critical to develop an understanding of how cocaine alters neurotransmission in the widespread projection areas of these and other monamine nuclei. Since 5-HT and opioids that inhibit GABA release from these terminals. Modulation at this synapse may be important in understanding mechanisms of drug reward and sensitization.

The first aim of this proposal is to determine the direct action of cocaine on the membrane properties of neurons (locus coeruleus (LC), substantia nigra (SN) and dorsal raphe (DR)) in dissociated cell culture. With isolated cells the direct action of cocaine on membrane properties and exogenously applied transmitters can be studied quantitatively since diffusion barriers and endogenous transmitters will be limited. Agonists can be applied in known concentration close to the cell. The effect that cocaine has on the kinetics of ligand activated conductances will be studied to determine if cocaine changes the affinity of the receptor for the transmitter. In addition the use of cultured cells permits whole cell recording with patch electrodes such that more control of the intracellular ion content and second messengers is gained. The proposed experiments are a natural progression of previous experiments in brain slices. The knowledge and experience gained by the work from LC and DR neurons in slices over the past 8 years will be directly applicable to work on isolated cells. The second aim is to determine the changes induced in monoamine neurons recorded in brain slices taken from animals previously treated with repeated injections of cocaine. Chronic cocaine is known to cause long term changes in behavior. The brain slice is particularly will suited for the study of these changes at the single cell level since synaptic connections are left intact and quantitative pharmacological methods can be applied to determine any changes in receptor sensitivity. The long term goal is to use the strengths of each; the brain slice and isolated cell preparations to study action of drugs of abuse at the cellular level. The acute and chronic effects of cocaine will first be studied in cells from relatively homogenous nuclei (LC, SN and DR). Knowledge gained from those relatively simple nuclei will be applied to more complex areas such as the ventral tegmental area, nucleus accumbens and prefrontal cortex. Studies from cells taken from animals which have self-administered cocaine may lead to an understanding of the changes that cause and result from cocaine abuse. Whole cell recordings will also eventually be used to study the changes that occur with other drugs of abuse such as opioids. For example, such recordings from LC neurons with patch electrodes are vital to determine the nature of the reduction in the opioid 'spare-receptor' population following chronic opioid treatment.

The specific aim is to culture dissociated neurons from the locus coeruleus (LC), substantia nigra (SN) and dorsal raphe (DR) and study of the action of cocaine on single isolated neurons. With isolated cells the direct action of cocaine on membrane properties and exogenously applied transmitters can be studied quantitatively since diffusion barriers and endogenous transmitters will be limited. Agonists can be applied in known concentration close to the cell. The effect that cocaine has on the kinetics of ligand activated conductances will be studied to determine if cocaine changes the affinity of the receptor for the transmitter. In addition, the use of cultured cells permits whole cell recording with patch electrodes such that more control of the intracellular ion content and second messengers is gained. The long-term goal is to use the strengths of each; the brain slice and isolated cell preparations to study action of drugs of abuse at the cellular level. In the work proposed here, the acute and chronic actions of cocaine will be determined in cells from relatively homogenous nuclei (LC, SN and DR). The knowledge gained in these simple nuclei will be applied to the action(s) of cocaine in more complex areas such as the ventral tegmental area, nucleus accumbens and prefrontal cortex. Studies from cells taken from animals which have self- administered cocaine may lead to an understanding of some of the changes which cause or result from chronic cocaine use. Whole cell recordings will also eventually be used to study the changes which occur with other drugs of abuse such as opioids. Such recordings from LC neurons with patch electrodes are vital to determine the nature of the reduction in the opioid 'spare-receptor' population following chronic opioid treatment. This award will afford the PI the time to learn and become competent with the tissue culture and whole cell recording methods. The proposed experiments are a natural progression of those which are in progress in the slice preparations. The knowledge and experience gained by the work carried out on LC and DR neurons in the slice over the past 8 years will be directly applicable to work on isolated cells. This coupled with the expertise of members of the Vollum Institute in recording from cultured neurons provide an optimistic setting from which the PI can learn and apply this knowledge to the understanding of cellular mechanisms of drug abuse.

In a classical series of experiments, Martin and coworkers described the responses of the chronic spinal dog to several classes of opiate compounds (1-2). From these responses he described three types of opiate receptor designated mu, kappa, and sigma. The sigma receptor was proposed to account for the response observed with the experimental drug, SKF10047. This benzomorphan opiate produced an increase in respiratory and heart rate, mydriasis, and a striking behavioral syndrome suggesting a "canine delirium". The sigma-binding site has now been shown to be a unique, non-opioid binding protein present in the central and peripheral nervous system. Compounds that have high affinity for this site such as haloperidol, phenothiazines and even SKF10047, have interactions with other receptors making it difficult to accurately assess the physiologic and pharmacologic role of the sigma-binding site. Recently several high affinity ligands have become available that are selective for the sigma-site. This should make it possible to study the cellular effects of the interaction between sigma-ligands and the binding site. In the present proposal the action of sigma-ligands on single neurons in the locus coeruleus and dorsal raphe will be studied using intracellular recording in brain slices. The action of sigma-ligands on resting properties, spontaneous activity, membrane currents, synaptic potentials and ligand activated currents will be studied. Studies of specific intrinsic and ligand activated membrane currents in isolation will be carried out with the single electrode voltage clamp. As there has been little work done with selective sigma-ligands at the cellular level, this study will lead to an understanding of the cellular actions of this class of psychotomimetic compounds. Equally important, the results may lead to a better understanding of the pathogenesis of and new therapies for schizophrenia.

The experiments in this proposal are aimed at uncovering adaptive changes in neuronal activity that result from chronic opioid treatment. Behaviorally it can be observed that a high degree of tolerance to and dependence on morphine develops with chronic use. It is curious that, at the cellular and biochemical level, the degree of tolerance and dependence is orders of magnitude smaller. The disparity in the observations may suggest that there are multiple mechanisms arranged in series that could account for the high level of behavioral tolerance. Another possibility is that regulation of ion conductances other than those directly affected by opioids are altered with chronic treatment and they may account for the increased level of tolerance and dependence. Opioids activate potassium channels and/or the inactive calcium channels to perturb cell excitability and function. In the continued presence of opioids, tolerance develops to these direct actions of opioids. There are, in addition, other adaptive changes in cell function and excitability not directly related to the specific ion channels gated by opioids. The regulation of those channels 'indirectly' gated by opioids is the subject of this proposal. Intracellular and whole cell recordings from neurons in the locus coeruleus and substantia gelatinosa of the spinal trigeminal nucleus will be made in brain slice preparations. With these two preparations, two opioid mediated responses will be investigated, the increase in potassium conductance and the presynaptic inhibition of glutamate release. One consistent observation made with chronic opioid treatment is an increase in the basal and stimulated level of cAMP. In this proposal, the effects of cAMP-dependent processes on transmitter release and ion conductances gated by voltage, transmitters and G- proteins will be identified and characterized. These cAMP-dependent processes will be studied after acute application (min or hours) of opioid agonists and in animals treated chronically (days) with morphine. By investigating the alteration in the regulation of ion conductances not directly affected by opioids, it may be possible to clarify the mechanisms by which the high levels of tolerance found at the behavioral level are generated.

The specific aim is to culture dissociated neurons from the locus coeruleus (LC), substantia nigra (SN) and dorsal raphe (DR) and study of the action of cocaine on single isolated neurons. With isolated cells the direct action of cocaine on membrane properties and exogenously applied transmitters can be studied quantitatively since diffusion barriers and endogenous transmitters will be limited. Agonists can be applied in known concentration close to the cell. The effect that cocaine has on the kinetics of ligand activated conductances will be studied to determine if cocaine changes the affinity of the receptor for the transmitter. In addition, the use of cultured cells permits whole cell recording with patch electrodes such that more control of the intracellular ion content and second messengers is gained. The long-term goal is to use the strengths of each; the brain slice and isolated cell preparations to study action of drugs of abuse at the cellular level. In the work proposed here, the acute and chronic actions of cocaine will be determined in cells from relatively homogenous nuclei (LC, SN and DR). The knowledge gained in these simple nuclei will be applied to the action(s) of cocaine in more complex areas such as the ventral tegmental area, nucleus accumbens and prefrontal cortex. Studies from cells taken from animals which have self- administered cocaine may lead to an understanding of some of the changes which cause or result from chronic cocaine use. Whole cell recordings will also eventually be used to study the changes which occur with other drugs of abuse such as opioids. Such recordings from LC neurons with patch electrodes are vital to determine the nature of the reduction in the opioid 'spare-receptor' population following chronic opioid treatment. This award will afford the PI the time to learn and become competent with the tissue culture and whole cell recording methods. The proposed experiments are a natural progression of those which are in progress in the slice preparations. The knowledge and experience gained by the work carried out on LC and DR neurons in the slice over the past 8 years will be directly applicable to work on isolated cells. This coupled with the expertise of members of the Vollum Institute in recording from cultured neurons provide an optimistic setting from which the PI can learn and apply this knowledge to the understanding of cellular mechanisms of drug abuse.

Chronic use of morphine results in tolerance to and dependence on the drug. One mechanism underlying cellular tolerance is an uncoupling of the opioid receptor from effects such that greater receptor occupancy is required to obtain a given response. This uncoupling mechanism has been studied using effectors that include; potassium and calcium conductances and the inhibition of adenylyl cyclase. A similar phenomenon is observed with acute opioid desensitization. Acute desensitization is thought to involve at least two linked processes, receptor uncoupling through an arresting dependent mechanism and removal of receptors from the plasma membrane through an internalization mechanism. Unlike more protracted forms of tolerance, acute desensitization is reversible within minutes and therefore easier to study in vitro. One goal of this proposal is to define the events that mediate the initiation and recovery from acute opioid desensitization. This goal has two parts, one is to characterize acute desensitization and determine the role of receptor internalization in that process. Given that receptor trafficking is an important form of receptor regulation, the second goal is to characterize how chronic morphine treatment alters desensitization and internalization of the opioid receptor. With a better understanding of the events that mediate desensitization and internalization the processes leading to long term tolerance may be more easily identified. The second goal is to identify post-synaptic cellular adaptations to chronic opioid treatment. These adaptations oppose the initial effect of opioid such that normal function is attained even in the continued presence of morphine. Thus adaptive mechanisms underlie an important form of tolerance. Two cell types will be examined the neurons in the locus coeruleus and interneurons of the VTA. There is an extensive knowledge of opioid actions in these areas, however, the identification and characterization of a post-synaptic adaptive mechanism studied in isolation has yet to be presented. Knowledge of alterations in regulation of ion channels during withdrawal form morphine may help in the development of more efficient protocols for the prevention of relapse to drug use.

DESCRIPTION (provided by applicant): Synaptic transmission mediated by monoamines is terminated by cocaine-sensitive transporters. The overall hypothesis is that adaptations in monoamine release result from acute and repeated treatments with cocaine. The aims of this proposal are to identify and characterize synaptic adaptations in the Ventral Tegmental Area (VTA) that result from acute and repeated cocaine treatments. One aim of this proposal is to completely characterize an inhibitory postsynaptic potential caused by the synaptic release of dopamine in the VTA. The hypothesis is that acute and repeated administration of cocaine will change pre- and postsynaptic elements that control this synaptic response. By recording the effects of synaptically released dopamine on dopamine neurons this study will be able to identify adaptive mechanisms at both pre- and postsynaptic sites. A second aim is to identify the mechanism by which intracellular calcium regulates the activity of D2- dopamine receptors. When internal calcium is low, the activity of D2-dopamine receptors is high. The hypothesis is that postsynaptic interactions between calcium and D2 receptors will be sensitive to cocaine given both acutely and with repeated treatments. The significance of the proposed studies is two fold. First, the regulation of the firing of dopamine cells by endogenous dopamine through D2-autoreceptors has been known for decades. The proposed experiments will address cellular aspects of that regulation and the effects of acute and chronic cocaine that were not possible previously. Second, the results of this study on both the release of dopamine and sensitivity of D2-dopamine receptors will be a starting point for the understanding of the adaptive changes in the physiology of the dopamine system following cocaine treatment.

DESCRIPTION (provided by applicant): The development of tolerance to opioids limits their effectiveness for the treatment of pain. A wide variety of opioids that are commonly used in the clinic for the treatment of pain and drug addiction have dramatically different pharmacological properties. One primary difference between agonists is the ability of different agonist to cause varying amounts of desensitization and internalization of mu opioid receptors. Agonists fall into three major groups, those that induce both desensitization and internalization, those that induce desensitization but not internalization and those that are ineffective at both. The role that desensitization and internalization have in the development to tolerance and dependence to opioids has been a controversial subject that has been studied in a variety of model systems. This exploratory proposal will develop the method of Flurescence Correlation Spectroscopy to investigate the mechanism that accounts for the varying actions of different opioid agonists on the mu opioid receptor. This is a relatively new method that uses optical measurements of the mobility of fluorescent molecules within a very small volume. These measurements are made at the plasma membrane, within intracellular compartments in various parts of the cell, including the cell body, dendrites, axons and terminals. The mobility of molecular is directly related to the molecular interactions such that agonist/receptor and receptor/effector associations will be identified. This study will use agonists that are fluorescent (aim 1) and fluorencescently tagged mu-opioid receptors (aim 2) in a model system, HEK293 cells that stably express epitope-tagged mu-opioid receptors. Electrophysiological, biochemical and imaging methods have been used to characterize many of the steps in opioid-receptor dependent signaling. This exploratory proposal will introduce a new way to investigate the mechanisms that underlie the different pharmacological profiles of opioid agonists. In the 2 years afforded this proposal, this method will be developed using a very well characterized model, the HEK293 cells. Once this method is established, the ultimate goal of applying it to study of primary neurons will be pursued. By gaining knowledge of the processes that are selectively affected by some agonists and not others, the mechanisms underlying the development of tolerance and dependence may be identified and applied to more effective treatment of chronic pain and addiction.

DESCRIPTION (provided by applicant): Dopamine plays a role in multiple physiological processes ranging from movement and reward to the regulation of hormonal balance. Dopamine receptor agonists or antagonists are used for the treatment of diseases such as schizophrenia, depression, attention deficit disorder, and Parkinson's disease. Recent work also indicates that the altered regulation of dopamine release induced by many drugs of abuse plays a critical role in early processes linked to early aspects of addiction. This there is a considerable understanding of the physiological role of dopamine in the central nervous system. There is likewise an enormous literature on molecular and biochemical aspects of dopamine signaling. Studies range from, the expression of the multiple is forms of dopamine receptors and second messenger cascades in cell lines to, biochemical and electrophysiological examination of the actions of dopamine and receptor agonists on neurons in various parts of the central nervous system. Thus the signaling cascades that are activated by dopamine receptors are well characterized. In spite of the wealth of knowledge of the dopamine system, little has been done on the actions of synaptically released dopamine at the cellular level. This hole in knowledge is not for the lack of effort, but lies in the fact that robust physiological detectors linked to dopamine synapses have been difficult to find in the major projection areas. This proposal uses recordings from dopamine neurons in brain slices from mouse to (1) define the inhibitory synaptic potential (IPSP) that results from the dendritic release of dopamine, (2) identify the presynaptic mechanism(s) and sites of dendritic dopamine release and (3) determine how the dendritic release of dopamine is altered following the induction of excitatory synaptic plasticity resulting from the treatment of animals with cocaine. The robust connection between the dendritic release of dopamine and the activation of an IPSC remains the best and probably only site at which dopamine transmission has been directly examined. The results of this study will therefore form a connection between knowledge at molecular and systems levels. The characterization of the synaptic mechanisms that determine rise and fall of extracellular dopamine in combination with a physiological response, will add considerably to the decades long search for an understanding of the role of dopamine in health and disease. PUBLIC HEALTH RELEVANCE: Dopamine agonists and antagonists are used clinically for the treatment of a number of diseases pointing out the importance of a complete understanding of the cellular and synaptic processes mediated by this important transmitter. The results from this study will define the cellular interactions between endogenously released dopamine and the receptors that detect the release of dopamine. From this work knowledge of dopamine-dependent transmission in many areas of the brain will be facilitated and lead to a better understanding of the disruptions in transmission in many diseases including addiction.

DESCRIPTION (provided by applicant): Mu-opioid receptors are widely distributed in the central and peripheral nervous systems. The action of opioids in the mesolimbic dopamine pathway activates the reward pathway(s) that are a key point in the multistep process leading to abuse and addiction. Opioid receptors are expressed in multiple parts of the mesolimbic system and the action of opioids in one area of this complex system may contribute to, but will surely not dominate all aspects of the rewarding properties of opioids. For the past 20 years the action of opioids in the ventral midbrain dopamine system rested on the GABA interneurons of the VTA. Inhibition of those interneurons was proposed to increase the activity of dopamine neurons through a disinhibition. It is now clear that the action of opioids in the ventral midbrain is more complex. There are a number of GABA neurons that innervate dopamine neurons and many of those neurons express opioid receptors. Each of these afferent pathways can now be manipulated selectively with the use of optogenetic tools such that a comprehensive picture of opioid action can be obtained. This proposal will identify the opioid sensitive GABA inputs to dopamine neurons in naive and morphine treated animals. In order for disinhibition to be functionally relevant, there has to be a substantial amount of inhibition initially. The hypothesisis that the source of inhibition is dependent on the state of the animal. Each inhibitory pathway will be recruited differentially depending on the behavioral state of the animal. The proposed experiments will use brain slice experiments to demonstrate that the relative role of different GABA afferent pathways changes during withdrawal from chronic treatment of animals with morphine. Knowledge that different afferent pathways mediate reward and withdrawal will allow a directed approach to limit both reward and withdrawal. PUBLIC HEALTH RELEVANCE: Opioids activate the mesolimbic reward pathway at multiple sites. The inhibition of GABA inputs to dopamine neurons is one important site of opioid action(s). This study will use selective activation of GABA inputs in slices from naive and morphine treated animals to determine which GABA pathway dominates inhibition of dopamine neurons. Knowledge of the inhibitory control of dopamine neurons will facilitate the development of protocols that will reduce the abuse liability of opioids.

DESCRIPTION (provided by applicant): This program for pre- and post-doctoral training supports the training of specialists who are able to conduct preclinical research at levels ranging from the molecular to the cognitive/clinical, on the biological mechanisms underlying the development, maintenance, and elimination of drug-seeking behavior. Thirty-two members of the graduate faculty of the Oregon Health & Science University serve as preceptors for postdoctoral research fellows and for Ph.D. students matriculating into basic science graduate programs in behavioral neuroscience, neuroscience, physiology and pharmacology, or biochemistry. Major research areas represent four levels. Some faculty members work primarily at the cellular/molecular level, using molecular biological, cell biological, and electron microscopic techniques. Other faculty work principally at the level of physiological, biochemical and pharmacological systems, using receptor binding, autoradiography, in vivo microdialysis and voltammetry, and electrophysiological techniques, and some work primarily in behavioral pharmacology and pharmacogenetics, using behavioral testing, intravenous drug self-administration, quantitative genetics and genetic mapping, as well as computer modeling techniques. Finally, some faculty work with human subjects, using cognitive testing and a variety of imaging techniques. Areas of extensive existing faculty collaboration include: studies of dopaminergic systems, ranging from molecular biology to behavior; extensive studies of genetic determinants of drug responses, at all levels from molecular to statistical gene mapping; and the study of learned and unlearned determinants of responses to drugs, particularly their rewarding effects and drug self- administration. Sensitivity, tolerance, and dependence/withdrawal phenomena for all major classes of drugs of abuse are under active investigation. Training includes firm curricular grounding in the basic sciences, specific pharmacological training in abused drugs, and extensive and continuous participation in research.

? DESCRIPTION (provided by applicant): The role of dopamine in health and disease has been investigated for decades. Dopamine receptor agonists or antagonists are used for the treatment diseases such as schizophrenia, depression, attention deficit disorder and Parkinson's disease. Recent work also indicates that the altered regulation of dopamine release induced by many drugs of abuse play a critical role in early processes linked to early aspects of addiction. Thus there is a considerable understanding of the physiological role of dopamine in the central nervous system. Recent work indicates that dopamine neurons that project of various areas in the CNS are remarkably heterogeneous. The heterogeneity is the result of variable intrinsic properties due to differential expression of ion channels and transmitter receptors. It is also clear that afferent inputs to dopamine neurons vary within the ventral midbrain. Thus experiments from dopamine neurons with identified projections is a necessary step in obtaining an understanding of the mechanisms that regulate dopamine release in functionally different areas of the CNS. This proposal uses recordings from identified dopamine neurons in brain slices from mouse to (1) define the inhibitory synaptic potential (IPSP) that results from the dendritic release. (2) Identify the distribution of D2 receptors on the somato-dendritic membrane and relate that distribution to identified synapses distinguished by the presence of PSD95. (3) Determine how D2-receptor function and distribution are altered following treatment of animals with cocaine. The results of this study will form a connection between cocaine-induced plasticity at AMPA and D2 receptor dependent synaptic events. The characterization of these early events following treatment with cocaine will add considerably to the decades long search for an understanding of the role of dopamine in health and disease.

Brain functions are executed by intricately coordinated networks of neurons, whose modes of operation are highly sensitive to a constellation of neuromodulators. More specifically, neuromodulators such as dopamine, norepinephrine, serotonin, and acetylcholine exert dramatic control over global brain processes such as arousal, attention, emotion, or cognitive perception. Altered neuromodulator signaling has been linked to neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, depression and addiction. Similarly, opioid neuropeptides play important roles in the modulation of cognition and behavior. While the anatomical structures that produce neuromodulatory signals are well known, little is known about the spatial and temporal evolution of these signals in the innervated brain regions. This is because current measurement techniques, such as microdialysis or cyclic voltammetry, lack the spatial or temporal resolution (and often the molecular specificity) to resolve respective signals. This technical challenge has been a long-standing barrier to our understanding of how neuromodulation alters neural circuit function in order to influence behavior. To address this challenge, this project will develop, validate, and disseminate novel genetically encoded fluorescent proteins for large-scale optical measurement of monoamine neuromodulator and opioid neuropeptide signaling in behaving animals, by bringing together a multi-disciplinary team of investigators with unique and complementary expertise. These sensor proteins have the potential to revolutionize neuroscience in a way similar to genetically encoded indicators for calcium, glutamate, and voltage, which are now in widespread use. Combined with calcium and voltage imaging, neuromodulator sensors will reveal how these systems impinge on cellular and circuit function. In particular, proposed sensors will enable minimally invasive, high-resolution, long-term, and direct measurement of neuromodulator and neuropeptide signaling at synaptic, cellular, and system levels. Sensors for neuromodulatory signaling have remained elusive for a long time. Our team recently developed a first generation of genetically encoded indicators for serotonin (5-HT), norepinephrine (NE), and dopamine (DA) that can report nano- to micromolar concentration changes with high spatial and temporal resolution. Building on this work, the following specific aims are proposed: 1) Optimize and diversify genetically encoded sensors for the monoamines using computational modeling, directed evolution and high-throughput screening; 2) Develop and optimize genetically encoded sensors for opiate neuropeptides using novel protein scaffolds; and 3) Systematically validate the novel neuromodulator and neuropeptide sensors in acute brain slices and behaving animals. Together, this work will provide the neuroscience community with a wide range of well-characterized multi-color indicators for probing the functional role of neuromodulators and neuropeptides in regulating neural circuit function and behavior in both health and disease.

Opioid receptors are widely distributed in the central and peripheral nervous system and upon activation have manifold actions including; depression of respiration, activation of the reward pathway, disruption of normal gastrointestinal motility and analgesia. It is currently difficult to examine this widespread distribution in wild type animals. Antibodies against the opioid receptors are often very unreliable and the epitopes are most often intracellular such that detection in living tissue is not possible. The use of transgenic or knockin animals to identify receptors requires extensive characterization, can be limited by varied expression levels and has been limited to studies in mice. The goal of this proposal is to use a ligand directed approach to covalently label endogenous opioid receptors in living tissue. The guide compound, naltrexamine, is a non-selective opioid antagonist that has high affinity for all subtype opioid receptors. Naltrexamine will be coupled through a series of linkers, a reactive acyl imidazole and fluorescent ligand. Once bound to the receptor, the reactive acyl imidazole reacts with a lysine on the receptor and simultaneously releases the naltrexamine. Thus natrexamine is free to dissociate from the receptor, however the fluorescent ligand is left covalently bound to the receptor. Preliminary results indicate that this compound interacts very effectively with the mu-opioid receptor. Thus endogenous receptor are labeled and the labeling is not dependent on species. This compound and future refinements will be used areas of the CNS where opioid receptors are expressed in only a small population of neurons. This proposal will center on the VTA and Substantia Nigra in mouse and rat. Opioid receptor expressing neurons in living brain slices will be identified and whole cell recordings will be made. By targeting these cells a complete understanding of opioid action in this key area of the reward system will be possible. The results of this cell based study will address a long-standing question surrounding the mechanisms that underlie one aspect of the addictive properties of opioids.