Barry Waterhouse - US grants

An initial series of studies will investigate basic physiological actions of norephrine (NE) and serotonin (5-HT) in the somatosensory and visual areas of the rat cerebral cortex. The goal of these studies is to establish a basis for assessing noradrenergic and serotonergic function in two animal models of epilepsy; cortically kindled and genetically epilepsy prone (GEPR) rats. Furthermore, tests used to examine biogenic amine function can be employed to clarify the mode of anticonvulsant drug action at synaptic levels within central neuronal circuits. A combination of microiontophoretic techniques, stimulation of biogenic amine, visual and somatosensory afferent pathways and computer assisted analysis of peri-event histograms will be employed to quantitatively assess the effects of NE, 5-HT and several anti-epileptic agents on cerebrocortical neuronal responsiveness to synaptic inputs and putative transmitter substances. The primary concept to be tested is that NE and 5-HT exert modulatory influences on synaptic efficacy within the cerebral cortex. Specific studies in seizure prone animals will use the same protocols to assay for an alteration in cerebrocortical biogenic amine function which might correlate with increased susceptibility for convulsive episodes. Anticonvulsant drug effects on synaptically mediated and transmitter induced cortical neuronal responses will be compared with NE/5-HT actions to determine if these compounds share in common mechanisms to modify transmission of information through neocortical circuits. Additional studies will use the 14C-2-deoxy-D-glucose (2-DG) technique in conjunction with a computer-based neuroanatomical image analysis system to determine glucose utilization patterns in the brains of normal and seizure susceptible animals before and after presentation of seizure-inducing stimuli and anticonvulsant drug administration. The aim of these studies will be to correlate regional differences in glucose metabolism, particularly in the monoamine nucleii and their target structures in the CNS, with seizure susceptibility and anti-epileptic drug efficacy. The proposed research will contribute to a basic understanding of noradrenergic and serotonergic function in the cerebral cortex and clarify the role of these biogenic amines in seizure disorders and mechanisms of anticonvulsant drug action.

An initial series of studies will investigate basic physiological actions of norepinephrine (NE) and serotonin (5-HT) in the somatosensory and visual areas of the rat cerebral cortex. The goal of these studies is to establish a basis for assessing noradrenergic and serotonergic function in two animal models of epilepsy; cortically kindled and genetically epilepsy prone (GEPR) rats. Furthermore, tests used to examine biogenic amine function can be employed to clarify the mode of anticonvulsant drug action at synaptic levels within central neuronal circuits. In some cases, these same issues will be addressed using the cerebellar Purkinje cell as a model system for electrophysiological and neuropharmacological study. A combination of microiontophoretic techniques, stimulation of biogenic amine and synaptic afferent pathways and computer assisted analysis of peri-event histograms will be employed to quantitatively assess the effects of NE, 5-HT and several anti-epileptic agents on cerebellar and cerebrocortical neuronal responsiveness to synaptic inputs and putative transmitter substances. The primary concept to be tested is that NE and 5-HT exert modulatory influences on synaptic efficacy within target areas of the CNS. Specific studies in seizure prone animals will use the same protocols to assay for an alteration in cerebrocortical biogenic amine function which might correlate with increased susceptibility for convulsive episodes. Anticonvulsant drug effects on synaptically mediated and transmitter induced neuronal responses will be compared with NE/5-HT actions to determine if these compounds share in common, mechanisms to modify transmission of information through local brain circuits. Major new experimental strategies that have been developed include investigations of monoamine and anticonvulsant drug actions in awake, behaving animals and brain tissue slice preparations. The goal of studies in awake animals is to examine NE/5HT and drug effects under the most physiologically relevant conditions. In vitro studies employing extra and intracellular recording techniques offer the promise of revealing the mechanisms associated with monoamine and drug effects that are observed in vivo. Overall, the proposed research will contribute to a basic understanding of noradrenergic and serotonergic function in the cerebral cortex and cerebellum and clarify the role of these biogenic amines in seizure disorders and mechanisms of anticonvulsant drug action.

A major consequence of cocaine self-administration is heightened sensory perception, an action which most likely contributes to an overall positive drug experience. Despite the likelihood that this effect adds to cocaine's desirability as a recreational compound and plays a supporting role in drug craving and abuse potential, few studies have investigated the neural substrates underlying cocaine's influence on sensory information processing. The fundamental question to be addressed in the proposed project is how cocaine affects the transmission of afferent sensory signals to a primary sensory circuit within the neocortex. These experiments will be conducted in intact, anesthetized rats and will employ combinations of single cell extracellular recording, systemic and local (microiontophoretic or micropressure) methods of drug administration, activation of cortical afferent pathways, procedures for depletion of central monoamines and computer assisted analysis of peri-event histograms to evaluate interactions between cocaine and somatosensory cortical neuronal responses to synaptic inputs or putative transmitter substances. The hypothesis to be tested is that because of its well established effects on monoaminergic systems which project extensively to sensory areas of the brain including those in the cerebral cortex and because of the modulatory actions of these systems on sensory cortical function, cocaine should exert monoamine-like changes in cortical neuronal responsiveness to afferent sensory signals. In preliminary studies cocaine has already been shown to produce noradrener- gic- like facilitating effects on cortical unit responses to thalamocortical synaptic inputs and glutamate. After characterizing the effects of systemically-administered cocaine on somatosensory unit responses to synaptic stimuli, three specific issues will be addressed regarding the neural substrates responsible for these actions: 1) the cortical or sub-cortical site of these actions, 2.) their dependence on endogenous monoamines and 3.) their pharmacological specificity with respect to other cocaine-like substances. The goal of this work will be to define the actions of cocaine at the synaptic level within a monoaminergically-innervated sensory circuit of the mammalian brain. Such studies win provide much needed information concerning the physiological basis of the drug's "desirable" effects on sensory information processing and as such may lead to new strategies for treating and preventing cocaine addiction.

Many neuroanatomical studies have demonstrated that sensory areas of the mammalian brain receive a substantial noradrenergic innervation from the brainstem locus coeruleus (LC). Other work has shown that cells in the LC increase their tonic level of firing with arousal and discharge phasically in response to novel or behaviorally relevant sensory stimuli. These findings have prompted the suggestion that output from the LC plays an important role in regulating the transfer of sensory information through neural circuits according to changing behavioral contingencies. Many studies have used local methods of drug application to identity the cellular actions of exogenous norepinephrine (NE) in sensory circuits; however, there have been no detailed investigations to assess the impact of synaptically released NE on the signal processing capabilities of sensory pathways. Our fundamental hypothesis is that the LC-NE system regulates signal transmission along sensory pathways via anatomically specific efferent projections and physiologically selective influences on response properties of individual neurons in sensory circuits. In order to test this idea the current proposal establishes three major goals: 1) determine the organization of the LC efferent projection to target structures along the ascending somatosensory pathway in rat, 2) characterize the actions of NE on intrinsic and membrane response properties of morphologically and electrophysiologically identified sub- classes of neurons in rat "barrel field" cortex and 3) determine the effects of both tonic and phasic modes of LC output on response threshold and receptive field properties of rat "barrel field" cortical neurons. These studies will involve neuroanatomical tract tracing; extra- and intracellular recording from single neurons in anesthetized animals and in vitro tissue slice preparations, respectively; electrical and chemical stimulation of LC; mechanical stimulation of the mystacial vibrissae and computer based analysis of spike train data (in vivo studies) or membrane potential changes (in vitro studies). Completion of this work will not only clarity how the LC-NE system influences sensory stimulus coding properties of sensory neurons, but will also provide a foundation for predicting how sensory circuits would perform under behavioral conditions where output from the noradrenergic system is fluctuating. As such these studies will provide a much needed link between the cellular actions of NE and the proposed role of the noradrenergic system in regulating sensory responsiveness across behavioral states.

DESCRIPTION: (Adapted from applicant's abstract) Neuroanatomical studies have demonstrated that sensory areas of the mammalian brain receive a substantial noradrenergic innervation from the brainstem locus coeruleus (LC). Other work has shown that cells in the LC increase their tonic level of firing with arousal and discharge phasically in response to novel or behaviorally relevant sensory stimuli. Investigations from our laboratory as well as others have shown that local administration of norepinephrine (NE) can increase the magnitude of individual sensory neuron responses to synaptic stimuli. Collectively these findings have prompted the suggestion that output from the LC plays an important role in facilitating the transfer of sensory information through neural circuits according to changing behavioral contingencies. However, many questions remain concerning the precise way in which output from the LC impacts on the stimulus coding properties of single cells and ultimately how NE release influences the operation of ensembles of neurons engaged in common sensory functions. In addition there has recently been considerable interest in galanin, a neuroactive peptide which co-localizes with NE in sub-populations of LC neurons. Despite the potential for co-release of galanin at noradrenergic synapses there have been almost no studies to characterize its actions on cells in LC innervated circuits. Our fundamental hypothesis is that the LC efferent system regulates signal transmission along sensory pathways via: 1) anatomically and neurochemically specific efferent projections and 2) physiologically selective influences on response properties of individual neurons in sensory circuits. In order to test these ideas the current proposal establishes 3 major goals: 1) determine the organization of galaninergic projections from LC to the ascending trigeminal somatosensory pathway in rat, 2) determine the individual and combined effects of putative LC transmitters/modulators on intrinsic membrane and cellular response properties of identified neurons in rat barrel field cortex and 3) determine the effects of phasic vs tonic activation of LC on response threshold and receptive field properties of rat barrel field cortical neurons. The studies will involve retrograde tract tracing, immunohistochemistry, extra- and intracellular recording from single neurons in anesthetized animals and tissue slice preparations, electrical and chemical activation of LC, mechanical stimulation of the mystacial vibrissae and computer-based analysis of spike train data (in vivo studies) or membrane potential changes (in vitro studies). Completion of this work will not only clarify how the LC efferent system influences sensory stimulus coding properties of sensory neurons, but will also provide a foundation for predicting how sensory circuits would perform under behavior conditions where output from the LC is fluctuating. As such these studies will provide a much needed link between cellular/membrane studies of LC-NE attributes and the proposed role of the LC efferent system in regulating perceptual processes across behavioral states.

DESCRIPTION (provided by applicant): Attention deficit, hyperactive disorder (ADHD) is a childhood cognitive disorder characterized by inattentiveness and/or hyperactivity and impulsiveness. The most effective treatment for ADHD is chronic low dose administration of amphetamine (AMPH)-like stimulants such as methylphenidate. All of these agents have prominent effects on monoaminergic neurotransmission yet little is known regarding the specific mechanism(s) through which they exert their therapeutic action in ADHD. Almost all of our information concerning AMPH-like stimulants derives from drug abuse studies which employ doses far in excess of those used clinically. Furthermore, there are profound dose and drug-specific differences in AMPH-like stimulant actions on monoaminergic neurotransmission. The central tenet of the proposal is that it is inappropriate to postulate therapeutic mechanisms of action for AMPH-like stimulants on the basis of animal studies of drug abuse. Moreover, a better understanding of the therapeutic actions of these drugs is needed in order to develop pharmacological treatments that do not possess the negative properties of AMPH-like stimulants (e.g. unwanted behavioral side effects, long term toxicities, abuse potential). The goal of the proposed study is to develop methodologies for evaluating the effects of therapeutically-relevant doses of methylphenidate on: 1) catecholaminergic neurotrasmission, 2) the impact of acute and chronic low dose stimulants on sensory processing, sensory detection and attention, 3) the receptor mechanism involved in stimulant-induced alterations in sensory processing, sensory detection and attention, and 4) the long-term consequences of low dose stimulants on a variety of physiological and behavioral processes in developing animals. The major techniques to be employed here include gas chromotography for determining plasma levels of methylphenidate, microdialysis for determining plasma levels of methylphenidate, microdialysis for determining catecholamine levels in specified brain regions, EEG and EMG for identifying behavioral states of arousal, multi-channel many neuron recording in cerebral cortex, thalamus and locus coeruleus for characterizing patterns of neural discharge in awake behaving rats, and computer based analysis of many neuron spike train data. These studies will fill a significant gap in our understanding of therapeutic actions of AMPH-like stimulants in ADHD.

DESCRIPTION (provided by applicant): The goal of the proposed project is to investigate interactions between the central noradrenergic system and systemically administered methylphenidate (MPH) - RitalinR in juvenile, adult and aged rats. This amphetamine-like psychostimulant has been the drug of choice for treating the core symptoms (inattention, distractibility, impulsivity, hyperactivity) of attention deficit hyperactivity disorder (ADHD) for more than 20 years and is now gaining notoriety for its off-label use as a cognitive enhancer in healthy subjects. Despite a long history of prescription use and more recent illicit appeal, the neural circuit mechanisms responsible for methylphenidate's effects on sensory signal processing and cognition have not been elucidated. For example, it is well known that MPH blocks reuptake of synaptically released norepinephrine (NE) and dopamine in the brain but these biochemical actions do not provide a clear physiological explanation for the drug's efficacy in either normal individuals or ADHD patients. In this context there have been major advances in our understanding of the cellular/membrane actions of NE and dopamine on target neurons in the brain, yet we still do not fully comprehend how systemically administered agents interact with noradrenergic and dopaminergic systems to bring about changes in neuronal function, signal transfer, and behavior in intact animals. Previously funded and current work focuses on the endogenous NE system. The theme is to better understand specific dimensions of central noradrenergic function in the context of low dose psychostimulant drug action. The project employs a variety of experimental approaches including: 1) measurement of drug levels in blood plasma following systemic MPH administration, 2) stereological analysis of the distribution and density of noradrenergic profiles across various brain regions, 3) microdialysis and high pressure liquid chromatography with electrochemical detection (HPLC-EC) of NE release in sensory and cognitive brain areas before and after drug administration, 4) multi-channel, multi-neuron recording at relay sites along the somatosensory pathway in waking or anesthetized rats before and after drug, and 5) activation of the locus coeruleus-noradrenergic efferent pathway in waking or anesthetized animals. Protocols will be carried out primarily in adult animals but also in prepubertal juvenile and aged rats. The overall goal is to establish a link between the actions of low dose MPH and operation of the endogenous central noradrenergic transmitter system within sensory and cognitive brain circuits of juvenile, adult, and aged rats. Completion of this work will not only further our understanding of LC noradrenergic system function and psychostimulant drug action but will also provide insight regarding the pathology of ADHD and the motivation for off-label use of this class of drugs to promote wakefulness and enhance cognition in healthy subjects. PUBLIC HEALTH RELEVANCE: The goal of the proposed project is to investigate interactions between the endogenous norepinephrine transmitter system in the brain and systemically administered methylphenidate (Ritalin), an amphetamine-like psychostimulant that is the drug of choice for treating ADHD and is gaining popularity among otherwise healthy individuals across the aging spectrum for its wake promoting and cognitive enhancing effects. Methylphenidate elevates norepinephrine and dopamine transmitter levels in cognitive and sensory regions of the brain, yet the basic physiological mechanisms through which it brings about its therapeutic and cognitive enhancing effects are unknown. A series of anatomical, neurochemical, and electrophysiological experiments will be used to establish a link between methylphenidate's actions and operation of the endogenous norepinephrine transmitter system within sensory and cognitive brain circuits of juvenile, adult, and aged rats.

DESCRIPTION (provided by applicant): The number of underrepresented minority students entering into medicine, nursing, and related health care disciplines is declining. To illustrate, in the past 5-6 years, the number of underrepresented minority students entering into our medical college has dropped by about 82% (from 50 in 1998 to 9 in 2002). This gradual decline in enrollment is not unique to our medical college, for it is observed in many institutions of higher education across the nation. A similar trend is also witnessed in graduate education programs. One key approach that could be adopted to reverse this trend is to involve underrepresented minority students in biomedical and behavioral research during the course of their undergraduate education. Drexel University College of Medicine (Drexel Med) has been educating students in biomedical sciences for over 150 years. Its recent merger with Drexel University, a respected leader in engineering and computer sciences, has unveiled yet additional opportunities. At Drexel Med, we have the required experience and the expertise to successfully initiate and implement summer research programs. We currently have three programs which offer short-term summer research training opportunities to local high school, undergraduate, and medical students. These successful programs serve as an archetype for initiating, in partnership with NHLBI, similar research opportunities for underrepresented minority students in undergraduate and graduate programs in various schools and colleges within Drexel University and at Cheyney University - the oldest black historical university in the country. Since the primary goal of NHLBI is to support research in the areas of cardiovascular, pulmonary, blood, and sleep disorders, minority students enrolled under this training grant will be assigned to one of the many (> 19) investigators in basic and clinical departments who are working in areas of similar interests. We believe that by providing such an opportunity to underrepresented minority students in undergraduate and graduate programs, we will not only enrich their academic experience but would also facilitate their entry and retention into health care related professional pathways. This outcome would meet the growing needs of our racially divergent communities and help alleviate the existing discord between the representation of minorities in the general populace as compared to that in the health care profession.

[unreadable] DESCRIPTION (provided by applicant): 3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is a popular "recreational" drug with considerable abuse liability including potential neurotoxicities directed toward the central serotonergic system. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of monoamine transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the "recreational" range. For example, MDMA users report that one of the major pleasurable outcomes of ecstasy self-administration is enhanced tactile sensation, but there is no explanation for how the drug produces this desirable and much sought after sensory experience. Because of the potential for long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The long-term goal of the proposed research is to better understand the neurophysiology underlying ecstasy's effects. The immediate goal of the present proposal is to develop and validate procedures for evaluating the impact of MDMA on sensory signal processing in the VPM thalamus of intact rats. The project employs plasma level analysis of drug concentrations, and electrophysiological determination of VPM cellular function in intact anesthetized or waking rats. Multi-channel, multi-neuron extracellular recording, systemic drug administration, activation of afferent trigeminal somatosensory pathways, and computer based analysis of spike train data are used to assess the impact of MDMA on sensory signal processing. A significant feature of this multi-dimensional approach is that drug effects will be determined at acute and chronic doses that: 1) approximate human self-administration regimens, 2) produce known plasma levels of the drug, and 3) elicit measurable changes in monoamine efflux within sensory circuits of the brain. Understanding the relationship between MDMA administration, monoamine transmitter efflux, and the operation of the somatosensory system will provide a basis for understanding the neurophysiological mechanisms underlying the drugs' effects on tactile sensory perception, in particular; and neural circuit functions, in general. A detailed knowledge of MDMA's effects on cellular and neural circuit function including its effects on sensory neurophysiology is essential in order to provide the public with accurate information regarding the risks associated with recreational use of this popular compound and its derivatives. Furthermore, once established, these methods will be used in future studies to characterize MDMA actions in other brain networks (prefrontal cortex, limbic) and more sophisticated behavioral assays (sensory discrimination, self-administration, craving and re-instatement) as a means of further clarifying its abuse liability. Waterhouse, Barry D. PROJECT NARRATIVE ()3,4-methylenedioxymethamphetamine (MDMA/ 'ecstasy') is an increasingly popular "recreational" drug that poses a significant threat to the nation's health because of its: 1) adverse acute and chronic effects on behavior and physiological functions, 2) neurotoxicity toward selected neurotransmitter systems in the brain, and 3) overall addictive potential. Although much attention has focused on MDMA's neurotoxic effects and its' ability to promote release of endogenous transmitters, little is known about how the drug affects the operation of neural circuits and brain function either at high neurotoxic doses or doses considered to be in the "recreational" range. Because of the potential for addiction and long term damage to the nervous system after MDMA ingestion, there is a pressing need to understand the neural substrates underlying the motivation for human self-administration of this agent. The goal of the proposed project is to develop and validate procedures that will help us better comprehend the neurophysiological basis for ecstasy's effects in human drug users. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this multi-investigator project is to develop an animal model of HIV neuropathology that can be used to assess: 1) cognitive function, 2) neuronal and non-neuronal degeneration in the prefrontal cortex and 3) electrophysiological properties of cells and circuits in prefrontal cortical networks. With the advent of improved combination antiretroviral therapy, HIV infection has been transformed from a fatal illness to a chronic manageable condition. This trend has resulted in an increasingly large population of aging individuals with prolonged exposure to HIV neurotoxins and to HIV therapeutic interventions. While there are excellent tissue culture models for studying the impact of HIV or HIV therapy on cellular processes, the options for in vivo investigation of the effects o HIV infection or chronic antiretroviral therapy are more limited, particularly as they relate to th aging brain. The ideal model for investigating such issues would provide the opportunity to examine and correlate cognitive performance with electrophysiological indices of neural function and neuropathology across the aging continuum with respect to onset of the HIV infection and progression of ensuing disease processes. The work outlined in this proposal will focus on CNS exposure to the HIV envelope protein gp120 in adult and aged rats and its impact on 1) performance of two prefrontal cortex-dependent behavioral tasks, 2) neuronal excitability and synaptic transmission in the prefrontal cortical circuitry and 3) the degree of neurotoxic insult t neuronal and non-neuronal cells in the prefrontal cortex. The most important aspect of this investigation is the development of an animal model that will have advantages for numerous additional in vivo studies focusing on the broad array of potential agents and mechanisms associated with HIV infection and its treatment, the time course of these events, and their impact on the aging brain. In particular this model will facilitate the identification and development of new targets and new compounds for therapeutic interventions in adult and aging HIV/AIDS patients. Across all inquiries, the model will validate the findings of in vitro tissue culture studies and their relevance to normative functions in the intact central nervous system.

DESCRIPTION (provided by applicant): The primary goal of this project is to determine the anatomical organization, molecular phenotype, and electrophysiological properties of locus coeruleus (LC) neurons that innervate sub-regions of the rat prefrontal and motor cortex. The prefrontal cortex (PFC) including its sub-regions the orbitofrontal cortex (OFC), medial prefrontal cortex (mPFC), and anterior cingulate cortex (ACC), and the primary motor cortex (M1) are innervated by the LC and subject to the modulatory actions of its primary transmitter, norepinephrine (NE). Each of the PFC sub-regions mediates distinct aspects of executive function that are compromised in many neuropsychiatric disorders including ADHD, PTSD, and schizophrenia. Likewise, the NE system has been implicated in the control of many PFC-dependent behavioral functions including flexible and sustained attention. The first aim of the proposed research will include retrograde tracing studies and immunohistochemical procedures to identify and characterize the subsets of NE-containing neurons within the LC that project to each PFC sub-region. In this and all subsequent aims M1 will serve as a non-cognitive brain region for comparison. The second aim of the project will determine the molecular complement of each class of projection cells by examining their transcriptomes and their expression levels of selected genes that are critical for noradrenergic function using combinations of laser micro-dissection, RNA microarray analysis and qRT-PCR. The third aim will profile individual LC-PFC and M1 projection cells on the basis of membrane properties, synaptic responsiveness, and sensitivity to drug activation using whole cell patch clamp techniques. These studies will provide a more complete understanding of the anatomical, molecular, and physiological attributes of the LC-cortical projection and challenge the longstanding notion that the LC is a functionally homogeneous cluster of NE-containing neurons that exert uniform modulatory effects across all LC- noradrenergic terminal fields, simultaneously. Instead, the general working hypothesis is that the LC-NE system operates by heterogeneous and asynchronous modulation of modality-specific terminal fields. This new theoretical construct has far reaching implications for our understanding of normal brain function and treatment of many neuropsychiatric disorders that involve noradrenergic and prefrontal cortical dysfunction.