Gary Aston-Jones - US grants

Experiments are proposed to study the effects of experimental stroke and administration of antidepressant drugs on neurotransmission in the noradrenergic-locus coeruleus (NE LC) system. Recordings from single NE-LC neurons in anesthetized rats will examine the effects of middle cerebral artery ligation and acute or chronic administration of desipramine or phenelzine, on the soma excitability, responsiveness to noxious sensory stimuli, and post-excitatory inhibition of these cells. Similar recordings in unanesthetized behaving rats will determine the effects of these treatments on the spontaneous discharge of NE-LC neurons during naturally occurring behaviors and stages of the sleep-waking cycle, as well as their responsiveness to non-noxious sensory stimuli. Electrical stimulation of locus coeruleus, or iontophoresis of norepinephrine, will be used to test the effects of these same treatments on the postsynaptic responsiveness of cells in the parietal cortex and hippocampus to activity in the NE-LC system. The long-term objectives of this research are to determine the effects of stroke and of antidepressant treatment on neurotransmission in the NE-LC system by examining changes induced in both pre- and postsynaptic elements. Such data will be the first of their kind in examining stroke, and some of the first on antidepressants and the NE-LC system. These experiments may provide insights into mechanisms involved in affective disorders that often follow stroke, and further elucidate the role of the NE-LC system in the clinical effectiveness of antidepressant treatment of such disorders.

Experiments are proposed to study the effects of acute and chronic administration of alcohol, as well as withdrawal from chronic alcohol exposure, on neurotransmission in the noradrenergic-locus coeruleus (NE-LC) system. Recordings from single NE-LC neurons in anesthetized rats will examine the effects of various alcohol manipulations on the fiber conduction velocity, soma excitability, responsiveness to noxious sensory stimuli, and post-excitation inhibition of these cells. Similar records in unanesthetized behaving rats will determine the effects of these alcohol treatments on the responsiveness of NE-LC neurons to non-noxious sensory stimuli, as well as their spontaneous discharge during naturally occurring behaviors. Electrical stimulation of locus coeruleus, or iontophoresis of norepinephrine, will be used to test the effects of these same alcohol treatments on the postsynaptic responsiveness of cells in the cerebrocortex to activity in the NE-LC system. The long-term objectives of this research are to determine the effects of alcohol exposure on neurotransmission in the NE-LC system by examining changes induced in both pre- and postsynaptic elements. Such data will be valuable additions to the present sparse knowledge of the effects of alcohol on the physiology of this pervasive brain system. These experiments may provide insights into mechanisms involved in alcohol intoxication, addiction and withdrawal phenomena, and further elucidate the role of the NE-LC system in addiction in general.

DESCRIPTION (applicant's abstract): The long term goal of this project is to elucidate the mechanisms that regulate functions of the noradrenergic locus coeruleus (LC) system. Our previous results defined afferents to the LC nucleus, and characterized LC dendrites that extend into specific extranuclear zones. However, these findings also generated new questions: (i) what are the inputs to extranuclear LC dendrites, (ii) what circuits are LC afferents a part of, and (iii) what LC output functions are regulated by these afferent circuits? The proposed experiments will answer these difficult but important questions using new anatomical technology and electrophysiology. LC neurons have extensive extranuclear dendrites, and the zones containing these distal dendrites receive numerous inputs that do not innervate the LC nuclear core. However, it has been difficult to identify which of these afferents terminate on LC dendrites vs. other elements in this region. We will retrogradely label afferents that specifically innervate extranuclear LC dendrites using the recently developed transsynaptic tract-tracer, pseudorabies virus (PRV). Dendritic afferents will be confirmed by ultrastructural analyses, and their impact on LC neuronal activity will be determined. In addition to direct afferents to LC neurons, it is important to determine inputs to these direct afferents, and thereby identify circuits that regulate LC function. We will map indirect afferents to the LC in a detailed time-course study of transsynaptic transport of PRV. For prominent indirect afferents, the relays to the LC will be identified and the influence of these afferent circuits on LC activity will be determined. Our preliminary results indicate that the suprachiasmatic nucleus (SCN) is a prominent indirect input to the LC. The SCN is the brain's circadian pacemaker, and controls among other rhythms circadian properties of sleep and waking. As the LC has long been implicated in arousal, we hypothesize that the SCN-LC circuit is a key neural substrate linking circadian and sleep-waking processes. We will test this hypothesis by manipulating SCN activity, recording the effects on EEG arousal, and testing the role of the LC and associated relay nuclei in the effects obtained. This will be the first analysis of a neural link between circadian and arousal processes. These studies will extend our analysis of afferent control of LC function to identify inputs to distal LC dendrites and circuits that regulate LC activity. They will also provide the first demonstration of afferent circuit regulation of an important LC output function, cortical arousal.

This proposal is a request for funds to obtain a transmission electron microscope. The major users of the instrument have proposed four main projects which require the use of the instrument. First, the morphology of the neuromuscular junction and synaptosomes during age-associated changes will be quantified. Second, the frequency of myelinated monoaminergic nerve fibers in rat and monkey central nervous systems will be determined to serve as a baseline for comparisons between species, over time and after experimental manipulations. Third, quantitative determination of cholinergic axons in several regions of the brain, together with quantitation of the innervation of several connections between areas within the brain will be determined. Lastly, a quantitative study of the morphology of regenerating peripheral nerve and nerve muscle development during ontogeny has been proposed.

Despite a great deal of evidence that the noradrenergic locus coeruleus (LC) system is a key brain area involved in opiate abuse and withdrawal, and extensive knowledge concerning the actions of opiates on LC membrane properties, the effects and related mechanisms for opiates and opiate withdrawal on this nucleus in the intact, unanesthetized brain are poorly understood. Recent insights into afferent regulation of LC open the way for new investigations of opiate actions on this preeminent noradrenergic system. Experiments are proposed to study the effects of opiates on discharge of LC neurons and their major afferents, paragigantocellularis (PGI) and prepositus hypoglossi (PrH), in unanesthetized behaving rats. Effects of acute and chronic administration of morphine, and morphine withdrawal, will determined for spontaneous and sensory-evoked activity of unambiguous noradrenergic LC neurons in behaving rats. Recordings of impulse activity from PGI or PrH neurons antidromically identified as projecting to LC will document the effects of iontophoretically applied selective opiate agonists, naloxone and dextrorphan in naive and in chronically morphine treated, anesthetized subjects. In unanesthetized animals, the normative discharge characteristics of similarly identified LC-projecting PGI and PrH neurons will be determined, as well as their response to systemic acute and chronic morphine and morphine withdrawal. Possible morphine-induced changes in the excitability of afferent terminals in LC will also be examined in these animals. Responses of LC neurons to electrical stimulation of their major afferents, and the ability of amino acid antagonists to block afferent influences, will be established in waking animals. In addition, effects of locally microinfused, selective opiate agonists and antagonists on regulation of LC from these major afferents in naive and chronically morphine pretreated, unanesthetized behaving animals will be determined. These experiments will test the hypotheses that (i) opiates may have substantial impact on LC activity through altered afferent regulation of these cells, and (ii) hyperactivity of LC neurons during opiate withdrawal is caused by increased excitation from PGI, the major afferent to LC. These studies will set the stage for future experiments in which amino acid antagonists will be tested for their ability to block hyperactivity of LC during morphine withdrawal. The ability of excitatory amino acid antagonists to prevent or reverse withdrawal-induced hyperactivity in LC would be of particular clinical interest, calling for the design of such drugs for use in human opiate withdrawal programs. The long term goals of this research are to determine the effects of morphine on functionally important discharge properties of unambiguous noradrenergic LC neurons in physiologically intact animals, and to determine the sites and mechanisms by which opiates affect these cells. Such data will resolve conflicts and fill gaps in the present literature concerning the effects of this intravenously abused drug on functions of the ubiquitous noradrenergic brain system, and clarify the role of this system in opiate abuse and addiction.

Long-term Research: Reticulospiral Function in the Lamprey This award recommendation is made under the Program for Long and Medium-Term Research at Foreign Centers of Excellence. The program is designed to enable young U.S. scientists and engineers to conduct long-term research abroad at research institutions of proven excellence. Awards provide opportunities for the conduct of joint research and the utilization of unique or complementary facilities, expertise and experimental conditions in foreign countries. The award will support Dr. Vincent Pieribone, Hahnemann Medical College and Hospital, Philadelphia, for a fifteen-month research visit with Dr. Tomas Hokfelt, Karolinska Institute, Stockholm, Sweden. The award recommendation provides funds to cover, as appropriate, international travel, local travel abroad, stipend, dependents allowance, if applicable, and a flat administrative allowance of $250.00 for the U.S. home institution.

Barrington's nucleus is a collection of cells in the brainstem that projects to spinal cord neurons innervating the bladder. Based on lesion and stimulation studies, this nucleus has long been thought to be involved in the micturition reflex. Recently, Dr. Valentino noted that this nucleus contains one of the most dense clusters of corticotropin-releasing factor (CRF)-immunoreactive neurons in the brain. Since CRF is the primary brain hormone that initiates the stress response and that stress increases the synthesis of CRF in these neurons, she has proposed that Barrington's nucleus is involved in the regulation of autonomic function under stressful conditions. Dr. Valentino will systematically study this major CRF-containing brain nucleus and its possible role in stress. She will employ state-of-the-art anatomical methods to map the afferent and efferent connections of CRF-containing neurons of Barrington's nucleus in the brain. These studies will identify the neural circuitry by which stressors are able to communicate activity to this nucleus. In addition, Dr. Valentino along with her collaborator, Dr. Aston-Jones, will use electrophysiologic criteria to determine how different kinds of stressors alter activity of these CRF-containing neurons in this region. The results will not only provide new information about the function of Barrington's nucleus but may lead to a better understanding about how the brain integrates the multicomponent response to a stressor.

Previous studies indicated that the locus coeruleus-norepinephrine (LC-NE) system regulates responsiveness to stimuli, and the capacity to process information during stress. Our recent findings have provided several new insights into the role of the LC-NE system in vigilance and attention, some of which suggest important modifications of our previous ideas and those of others. Specifically, we have found that variations in LC activity in the behaving monkey correspond to marked changes in attentiveness. These results lead us to postulate a specific role for the LC in attentional processing. We propose that the LC regulates the stability/lability aspect of attention, denoted here as attentional lability. This dimension of attention rages from focused/selective attention (relatively non-distractible) to scanning/labile attention (easily distractible). The present proposal seeks to extend our recent observations to test this hypothesis and define LC's role in attention. The following studies are proposed: (1) We will record monkey LC neural activity during an attentional disengagement task, designed to measure the ability to change the focus of attention. This will allow analysis of LC's involvement in attentional lability. (2) Local microinjections of selective pharmacologic agents into the LC will be used to transiently and specifically inactivate or activate LC neurons during the attentional detachment task or a vigilance task. The effects of these manipulations on fluctuations in attention (measured by visual fixation performance) will be determined. Effects will also be discerned on behavioral responses in the two tasks. These experiments will test LC's causal role in focused and labile attention, and determine whether different levels of LC activity are sufficient or necessary for such attentional processes. (3) Environmental or cognitive stressors will be administered to determine their effect on LC activity in the behaving primate, and to determine the role of LC in mediating the effects of stress on attentional performance. (4) We will locally microinfuse pharmacologic agents into the LC to control impulse activity during task reversal and test the hypothesis that altered LC activity plays a critical role in the acquisition of stimulus significance. Thus, these experiments will extend our studies to examine the role of the LC in a specific type of learning. The proposed studies will examine in detail both the temporal association (via LC recordings) and functional dependency (via LC manipulations) between the brain noradrenergic LC system and attentional performance during normative as well as during stressful conditions. They will also be the first to examine the role of the primate LC in learning, and investigate the LC as a possible neural substrate for the linkage between attention and learning.

DESCRIPTION (adapted from applicant's abstract): The locus coeruleus- norepinephrine(LC-NE) system has long been implicated in arousal and alertness. This implies a relatively non-specific role for LC in cognitive function. However, the investigators' recent recordings from LC in the behaving monkey suggest a revision of this view. They have found that LC neurons respond to task-relevant stimuli in a selective, stimulus-specific manner, and that these cells exhibit two modes of activity corresponding to levels of performance in a visual discrimination task. Based on these findings, the investigators hypothesize that the LC helps to regulate the balance between selective vs. flexible behavior. They now have developed a computational model of LC and its influence on performance in cognitive tasks, which delineates mechanisms for such a function. This model predicts that the mode of LC activity influences the state of cortical function to promote either selective responding to task relevant stimuli or responsivity to a broader range of environmental stimuli, each of which may have adaptive value for behavior. Other of the investigators' results indicate that electronic coupling may be an important factor regulating the mode of LC activity. This proposal seeks to test these hypotheses, specifically in the context of tasks that involve visual spatial attention. The following studies are proposed: (1) The investigators will integrate our model of LC function into a model they have already developed of performance in a spatially-cued reaction time SCRT) task. (2) The investigators will record from monkey LC neurons during performance of the SCRT task, and analyze the relationship between LC activity and task performance, to test predictions made by the computational model. (3) The investigators will make local microinjections of selective pharmacologic agents into the monkey LC to transiently manipulate activity of, and electrotonic coupling among, LC neurons. This will test the causal role of LC in task performance, as well as the specific hypothesis that coupling among LC neurons is one means of regulating the mode of LC activity and the corresponding pattern of behavioral performance. (4) They will record visually responsive neurons in parietal cortex while recording and manipulating LC activity, to test mechanistic hypotheses concerning LC-NE influences on cortical function and task performance. The proposed program of research integrates computational modeling, neurophysiology, and behavioral studies to understand the functions of the LC-NE system, its interaction with cortical systems, and its regulation of the balance between task- directed behavior vs. responsivity to unexpected environmental stimuli.

DESCRIPTION: Applicant's Abstract Recent evidence from a variety of sources indicates that components of the 'extended amygdala' are importantly involved in opiate withdrawal (OW). This application seeks to extend our previous work on the noradrenergic locus coeruleus (LC) and OW by characterizing the role in OW of norepinephrine (NE) inputs to a key component of the extended amygdala, the bed nucleus of the stria terminalis (BNST). The BNST is the focus of this application because (I) it receives a very dense NE innervation, (ii) it is a key element of the extended amygdala that has been overlooked in studies of OW, and (iii) our recent data reveal that neurons in the BNST, and its NE afferents, are strongly responsive to OW. A set of coordinate anatomical and electrophysiological experiments are proposed that will characterize basic neurobiological attributes of NE in the BNST. Anatomical experiments will characterize the NE innervation of the medial and lateral subdivisions of the BNST, specifying innervation patterns and fiber morphologies. Using tract-tracing combined with immunohistochemistry, the sources of NE input to the different BNST subdivisions will also be identified. Electrophysiological studies will then confirm and characterize the inputs identified anatomically. The influence of the different NE afferents on BNST impulse activity will be determined, and the adrenoceptors involved in mediating responses will be identified. These studies will be some of the first to systematically investigate basic attributes of this dense noradrenergic projection to the extended amygdala. The role of this NE target area in OW will also be examined from the cellular to the behavioral level. We will determine the electrophysiological response of BNST neurons to OW; preliminary evidence for this application indicates that these cells will be strongly activated. The role of NE inputs from the different source cell groups in this OW response will be determined, and the adrenoceptor involved will be identified. Finally, in behavioral experiments the contribution of the NE-BNST synapse to somatic and aversive responses to OW will be determined. In particular, we will explore the role of the NE input to the BNST in conditioned withdrawal responses, a critical element in continued drug abuse and relapse. Together, the above experiments represent a comprehensive analysis of cellular substrates, and physiological and behavioral consequences, of the dense NE input to the BNST. These studies will provide much needed basic and withdrawal-related information on an important but neglected NE target in the extended amygdala.

We recently found that animals made dependent on morphine exhibit increased drug preference 5 weeks after withdrawal. These results reveal that prior prolonged drug exposure and withdrawal alters behavioral and neural responsivity to subsequent drug administration for a substantial period of time. This observation establishes a simple behavioral model of the well-established clinical observation that former addicts have a high liability for future relapse. Our goal is to identify the neural changes that underlie this long-term alteration of drug responsivity. We hypothesize that this increased drug preference after prior exposure and withdrawal is due at least in part to changes in pathways converging on the ventrolateral bed nucleus of the stria terminalis (vBNST). In particular, we hypothesize that norepinephrine innervation of the vBNST from the A2 neurons in the nucleus tractus solitarius, or corticotropin releasing hormone inputs from the amygdala or intrinsic BNST neurons, plays a role in potentiating activity of the vBNST in response to drug-associated stimuli. We recently found an excitatory amino acid pathway from the vBNST to the ventral tegmental area; this projection strongly activates dopaminergic neurons and may therefore play a pivotal role in expression of the enhanced drug preference. We propose a set of coordinated anatomical, neurophysiological and behavioral experiments to test these hypotheses. Results of these studies will provide important new insights into neural mechanisms underlying conditioned drug seeking and relapse in abstinent addicts, a major 3roblem in treatment and prolonged abstinence.

DESCRIPTION (provided by applicant): We recently found that rats made dependent on morphine show decreased learning and preference for food reward at both two and five weeks post-withdrawal. We also found that animals show increased preference for morphine-associated environmental cues during these same time periods after withdrawal. These place-conditioning paradigms provide a simple model of the dysregulation of reward processing and dysphoria that occurs during opiate abstinence. This dysregulation is generally believed to contribute to elevated preference or seeking for drugs and drug-related stimuli. Our goal is to identify the neural changes that underlie this long-term alteration of reward processing. Preliminary data revealed that neurons in the nucleus accumbens shell, lateral hypothalamus and basolateral amygdala alter their responsiveness to food- or morphine-conditioned stimuli in proportion to the amount of preference expressed. We hypothesize that changed neural function in these areas that regulate ventral tegmental area (VTA) dopamine neurons is critically involved in the associated shift in hedonic values. We further propose that protein kinase A function is altered in these VTA afferents during protracted withdrawal, resulting in compromised plasticity. This change in neural plasticity in the mesocorticolimbic dopamine system is proposed to underlie long term alterations in reward processing. A coordinate set of behavioral and anatomical studies is proposed to test these hypotheses. Together, these studies will identify neural substrates for altered reward processing and hedonic values following chronic drug exposure that may be critical in relapse during long-term abstinence.

3,4-Dihydroxyphenethylamine; 4-(2-Aminoethyl)-1,2-benzenediol; 8-Azabicyclo(3.2.1)octane-2-carboxylic acid, 3-(benzoyloxy)-8-methyl-, methyl ester, (1R-(exo,exo))-; Animals; Area; Arousal; Behavior; Behavioral; Brain; CNS plasticity; Cell Nucleus; Cells; Chemical Stimulation; Cocaine; Complex; Condition; Contin, MS; Cues; DA Neuron; Data; Dopamine; Dopamine neuron; Drug abuse; Drugs; Encephalon; Encephalons; Extinction; Extinction (Psychology); Face; Gelineau Syndrome; Glutamates; Goals; Hcrt protein; Hcrt/ORX; Hcrts/ORXs; Hydroxytyramine; Hypothalamic structure; Hypothalamus; Individual; Infumorph; Intervention; Intervention Strategies; Investigators; Kadian; L-Glutamate; Label; Lateral; Lead; Learning; Lesion; Location; Locus Coeruleus; MSir; Medial; Mediating; Medication; Mesencephalon; Methods; Microinjections; Mid-brain; Midbrain; Midbrain structure; Morphia; Morphinan-3,6-diol, 7,8-didehydro-4,5-epoxy-17-methyl- (5alpha,6alpha)-; Morphine; Narcolepsy; Narcoleptic Syndrome; Nerve Cells; Nerve Impulse Transmission; Nerve Transmission; Nerve Unit; Nervous; Nervous System, Brain; Neural Cell; Neurocyte; Neuronal Plasticity; Neuronal Transmission; Neurons; Neuropeptides; Neurotoxins; Nucleus; Nucleus Accumbens; Nucleus Pigmentosus Pontis; Oramorph; Oramorph SR; Paroxysmal Sleep; Pathway interactions; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Play; Prefrontal Cortex; Principal Investigator; Process; Programs (PT); Programs [Publication Type]; Public Health; Receptor Protein; Relapse; Research Personnel; Researchers; Response to stimulus physiology; Rewards; Role; Roxanol; SUBGP; Self Administration; Site; Specific qualifier value; Specified; Statex SR; Stimulation, Chemical; Stimulus; Stress; Structure of locus ceruleus; Subgroup; System; System, LOINC Axis 4; Testing; Ventral Tegmental Area; Work; abuse of drugs; abuses drugs; addiction; base; behavioral extinction; blue nucleus; brain cell; day; dopaminergic neuron; driving behavior; drug abstinence; drug/agent; experiment; experimental research; experimental study; facial; heavy metal Pb; heavy metal lead; hypocretin; hypocretin/orexin; hypocretins/orexins; hypothalamic; interventional strategy; locus ceruleus structure; neural; neural plasticity; neuronal; neuroplasticity; neurotoxicant; neurotransmission; novel; orexin; pathway; preference; programs; public health medicine (field); receptor; relating to nervous system; research study; response; reward circuitry; reward processing; social role; stimulus/response; ventral tegmentum

DESCRIPTION (provided by applicant): Recent advances in viral-mediated gene transfer have produced an exciting new toolset for neuroscience research. In particular, many studies in the last 10 years have used microinjection of viral vectors to insert genetic material into brain neurons in vivo. However, typically these viral manipulations have not been directed at a specific cell type, but rather viral infection has been targeted at all cells within the injection site. In addition, there have been almost no studies using such technology to explicitly modulate firing activity of neurons. We propose here to take advantage of cell-type specific viral expression technology coupled with new designer receptors to overcome these limitations. The goal of this collaborative proposal is to develop and validate a method to insert designer receptors into orexin neurons in brain in vivo, so that impulse activity in this important cell group can be selectively controlled by systemically administered compounds that are otherwise pharmacologically inert. Orexins have recently been found to be important in reward and addiction, as well as in arousal. Future applications of this new technology would include regulation of activity in orexin neurons selectively to better delineate their role in addiction. In addition, selective manipulation of impulse activity in cell- type specific neurons can be applied widely to other neurons, and will be a valuable new tool in behavioral and electrophysiological analyses of neural function. PUBLIC HEALTH RELEVANCE The proposed research is relevant to human health because it will develop new tools for more selective and specific manipulation of genes in brain neurons. These new tools not only will be important for basic research into the functions of specific brain neurons with consequences for new therapeutic development, but will also lead the way for new clinical approaches in gene therapy for a large number of disorders of the nervous system. Given the roles of the orexin system in addiction and in sleep disorders, the specific method proposed will lead to new ways to control orexin neuron activity for treating addiction or sleep disorders such as narcolepsy.

DESCRIPTION (provided by applicant): Response inhibition (RI) is central to executive control of behavior, and multiple lines of evidence indicate that the locus coeruleus-norepinephrine (LC-NE) system is involved in RI. Specific cortical areas are also strongly implicated in RI, including a major target of LC neurons in rat, the orbitofrontal cortex (OFC). The go-nogo (GNG) and stop signal (SS) tasks are used to examine the neural substrates of response-restraint and response-cancellation, respectively. Importantly, these tasks are nearly identical in humans and rats. Moreover, NE actions in frontal cortical areas of both rats and humans are involved in RI, as well as in RI deficits in attention deficit disorder/hyperactivity (ADHD) subjects. These findings afford us the opportunity to conduct translationally relevant studies to examine the specific role of the NE-LC system and OFC in RI. We will obtain unit recordings from rat LC and OFC neurons during GNG and SS tasks to identify their roles in RI. Specifically, we will test the hypothesis that phasic activation of LC neurons, and NE actions in OFC, are importantly involved in these measures of inhibitory control. The selective NE reuptake inhibitor, atomoxetine (ATM), has recently been found effective for improving RI in normal and ADHD individuals. We hypothesize that this might be due, at least in part, to effects of this compound on the activity profile of LC neurons as well as its effect on LC-NE input to OFC cells. We will test this by recording LC and OFC neurons during GNG or SS tasks following ATM administration. Finally, we will use novel viral transduction methods to express the photosensitive cation channel channelrhodopsin-2, or choride pump halorhodopsin-3, selectively in LC-NE neurons. We will phasically photo-activate or -inhibit these cells, or their terminals in OFC, at specific points in these tasks to test a causal role for the NE-LC system and OFC in RI function. These proposed studies will substantially advance our understanding of inhibitory control through novel analyses of the contribution of the LC-NE system and OFC to RI task performance. The results of these studies will also provide a new approach to the design of drugs to treat human disorders involving impaired RI, including ADHD and drug addiction. PUBLIC HEALTH RELEVANCE: Response inhibition (RI) is an important cognitive function, and many disorders include an inability to inhibit inappropriate behavioral responses (e.g., attention deficit/hyperactivity disorder, obsessive-compulsive disorder and drug addiction). The proposed experiments will define the roles of key brain neurons in RI. This will facilitate development of better treatments for behavioral disorders that include deficient RI.

DESCRIPTION (provided by applicant): The goal of this project is to establish an empirical foundation for measuring locus coeruleus (LC)- norepinephrine (NE) system effects in target networks using functional magnetic resonance imaging (fMRI). LC has been implicated in a variety of mental and degenerative disorders and therefore is an important target for pharmacotherapy. LC-NE neurons have widespread modulatory influences on brain function to regulate attention and modify the gain of sensory neurons, for example. However, it has been difficult to measure such global influences due to limitations in the ability to selectively activae LC-NE neurons while monitoring CNS network activity. It also is unclear how fMRI metrics characterize network effects of phasic and tonic patterns of LC activity that relate to different states of attention. This project seeks to surmount these limitations using innovative optogenetic methods to induce different patterns of activity specifically in LC-NE neurons during ultra high-field functional imaging in vivo. Our preliminary data demonstrate that we can photoactivate LC-NE neurons selectively with optogenetics, and simultaneously observe strong BOLD responses throughout the rat brain. Aim 1 is to fully develop an approach that will measure and distinguish effects of tonic vs phasic LC activation on large scale brain networks with fMRI. Aim 2 is to use fMRI to measure alterations in somatosensory neuronal responses to hindpaw stimulation induced by selective LC- NE activation. These studies will provide the first reliable and valid approaches for evaluating basic science and therapeutic questions about LC function with non-invasive imaging. PUBLIC HEALTH RELEVANCE: The ability to non-invasively measure effects of locus coeruleus (LC) activity on its global target networks using magnetic resonance imaging would advance our understanding of the role(s) for this important brain system in a host of mental functions and dysfunctions, and facilitate rationale development of new pharmacotherapies. This project uses selective optogenetic activation of LC-norepinephrine neurons and ultra high-field functional MRI (fMRI) to provide the first controlled estimates of LC effects on global network function, including during sensory stimulation. The results will establish the extent to which specific patterns of LC activity, resembling those implicated in attention and behavioral flexibility, affect brain networks in ways that can be observed in human subjects using fMRI.

The purpose of the Animal Core is to provide a consistent, uniform, and reliable source of animals instrumented and trained in cocaine self-administration for use in all of the projects of the NARC. Rats wlll be catheterized and trained to self-administer intravenous cocaine using a standardized and clinically relevant experimental paradigm. Subjects will then be directed towards individual NARC projects for brain tissue extraction, electrophysiology, or assessment of behavioral responding to systemic or site directed manipulations in accordance with the specific aims of each NARC project. The animal core in this renewal will expand to incorporate a transgenic TH::Cre rat colony that will provide transgenic rats to NARC projects with opsins expressed selectively in dopamine neurons. As part of providing optogenetic support for projects using TH:Cre rats, the Animal Core supports an optogenetics core containing the necessary equipment and expertise to allow all NARC optogenetic studies to proceed in a synergistic and competent manner. The primary objectives of the Animal Core core are: 1. Provide rats to all projects that satisfy specific criteria regarding experience in drug self-administration, and in the degree of withdrawal, extinction, and/or abstinence, as well as regarding intracranial injections of viral vectors. 2. Manage the distribution of rats to projects to insure that statistically relevant group sizes and group replications are made available to each project in a timely manner. 3. Provide trained technicians to efficiently generate experimental subjects using a balanced group design. 4. Maintain a colony of TH::Cre rats individually validated for Cre expression and provide the numbers of identified Cre+ and Cre- rats required across NARC projects. 5. Serve as a NARC training center for MUSC and offsite investigators who desire training in self- administration, jugular catheterization, operant conditioning methods, viral vector design, viral vector microinjections, optogenetic practices, and other related experimental procedures. 6. Provide the equipment, resources, and personnel for using optogenetics to manipulate specific neurons as appropriate across NARC projects. This includes maintaining a supply of virus expressing the various opsin transgenes needed by the NARC projects.

Relapse is a persistent problem in cocaine addiction, and many important aspects ofthe brain mechanisms involved remain unknown. One key area for cocaine relapse in the rat model is the medial prefrontal cortex (mPFC); in particular, mPFC interactions with the nucleus accumbens (NA) and ventral tegmental area (VTA) are critical for reinstatement of extinguished cocaine seeking. Projects 1 and 2 in this center focus on molecular and cellular mechanisms involved in the mPFC-to-NA pathway during extinction and reinstatement. Little is known about the activities of neurons in mPFC that project to NA, or that receive dopamine (DA) from VTA, during these cocaine behaviors. Here, we will use Fos labeling and unit recording during extinction and reinstatement to measure impulse activity of mPFC neurons identified as projecting to NA core or shell. We also will capitalize on TH::Cre rats and optogenetics methods recently implemented in our lab to determine the influence of endogenous DA release on impulse activity of prelimbic cortex neurons that project to NA core during extinction and reinstatement. Together, these studies will provide an overall map of NA-projecting mPFC neurons that are activated during cocaine behaviors, and also measure their impulse activities with respect to specific task stimuli and behaviors during exinction or reinstatement of cocaine seeking. These findings will provide a detailed circuit analysis of behavior-related activities in these key mPFC neurons that will be important information to extend results of molecular- and cellular-level studies in other projects of this center.