Bradley E. Alger - US grants

The major goal of this proposal is to elucidate the electrophysiological effects of phorbol esters on neurons in the mammalian hippocampus. Phorbol esters are potent tumor promoters and inflammatory agents which are of neurophysiological interest because they can substitute for an endogenous compound, diacylglycerol, in binding to, and activating, protein kinase C. Protein kinase C has been indirectly implicated in mediating the actions of several putative neurotransmitters, but its actual physiological role in the central nervous system in unknown. Protein kinase C may participate, via a new intracellular messenger system, in the control of neuronal excitability. The highest level of specific phorbol ester binding occurs in the brain and, within the brain, in hippocampus. Preliminary experiments have revealed that phorbol esters affect neuronal properties selectively and may block one, or more, potassium potentials important for regulating activity. Hippocampal neurons will be studied, both in the in vitro slice and in an acutely dissociated neuron preparation. Each preparation offers certain distinctive advantages. Using the in vitro slice, we will both determine the neuropharmacological properties of phorbol ester actions and attempt to identify the mechanisms underlying the effects observed thus far, using intracellular electrophysiological techniques. The isolated cell preparation will be used to refine the investigation using whole cell voltage-clamp and patch-clamp methods. The isolated cell also offers the opportunity for studying the effects of diacylglycerol and protein kinase C on ionic currents. Malfunction in the control of normal neuronal excitability is at the root of several neurological diseases, including epilepsy and Huntington's disease. Understanding the neuronal function of protein kinase C may reveal the intracellular mechanism of action of a class of neurotransmitters and shed light on the regulation of neuronal excitability.

The major goal of this proposal is to investigate the role of the polyphosphoinositide-protein-kinase-C (PI-PKC) second-messenger system in the mammalian brain using the hippocampus as a model system. A number of important functions have been suggested for this system, including regulation of calcium- and potassium- dependent currents, transmitter release and long-term potentiation. Until now the system has primarily been studied using phorbol esters, plant-derived tumor- promoting compounds that activate PKC. Of particular interest in this phase of the project is the role of endogenous activation of PKC by acetylcholine and its analogs. Little is known about physiological effects of neurotransmitter activation of the PI PKC system. Electrophysiological responses of hippocampal neurons to cholinergic agents will be studied with intracellular recording in the slice preparation. In acutely dissociated and tissue-cultured neurons, whole-cell voltage- and patch-clamp techniques will be used. Direct activation of PKC by synthetic diacylglycerols and phorbol esters, in addition to direct intracellular injection of purified PKC, will be used to define which ionic currents are affected and how they are affected. We will also study the responses to injection of the other product of polyphosphoinositide breakdown, inositol-1,4,5-triphosphate. A variety of the known and suspected consequences of PKC activation imply that it is involved in control of neuronal excitability under normal circumstances. Malfunction in excitability control is a major problem in several neurological diseases, including epilepsy, Parkinson's and Huntington's diseases. Deficits in cholinergic systems have been implicated in Alzheimer's disease. Defining the roles of the PI-PKC system in excitability control may not only lead to better understanding of the disease states, but also contribute to rational design of drug therapy.

voltage gated channel; membrane channels; synapses; cerebellum; hippocampus; neural inhibition; maleimides; gamma aminobutyrate; evoked potentials; neuropharmacology; pertussis toxin; neuronal transport; electrodes; Animalia;

The phosphatidylinositol-protein-kinase-C (PI-PKC) system is a ubiquitous second messenger system in the central nervous system. However, in contrast to the depth at which it is understood at the biochemical and molecular biological levels, there is still a paucity of information regarding its neurophysiological functions at the cellular and molecular levels. Glutamate, PKC, voltage-dependent Ca2+ channels (VDCCs) and Ca2+- dependent K+ after hyperpolarizations have all been implicated in a model of the establishment of memory traces, long-term potentiation (LTP), and in epileptic and neurodegenerative phenomena. The highest concentrations of PKC are found in the hippocampus, in which both the physiological and pathological events are known to occur. The ultimate goal of this project is to understand the role of the neurotransmitter-activated PI-PKC system in the control of neuronal excitability using state-of-the-art electrophysiological techniques in several in vitro hippocampal neuron preparations: slices, tissue-culture and acute isolation. The interrelationships among glutamate, PKC, VDCCs and the AHP have not bee clarified. This project seeks to provide this clarification using whole- cell voltage clamp and patch clamp techniques, at two levels: the nature of the second messenger control exerted by glutamate and the regulation of VDCCs and AHP channels by PKC. Investigation of these general issues will take place by testing specific hypotheses that have been developed from past work on this project. The hypotheses are: 1) that high-voltage- activated hippocampal Ca2+ channels are regulated by the action of a complementary kinase-phosphatase system involving PKC and a protein phosphatase, 2) that glutamate depresses Ca2+ currents via PKC activation and 3) that the hippocampal AHP current is regulated by PKC at the level of the single AHP channel. These hypotheses make explicit predictions and are susceptible to clear tests with voltage- and patch-clamp methods. The outcome of these tests will provide valuable information as to the functional role of the PI-PKC system, and, since ion channels and neurotransmitters to be studied have been implicated in a variety of neurological disease states, particularly epilepsy, to the understanding of, and ability to influence, these conditions as well.

The sodium pump (Na+/K+-ATPase) is nearly ubiquitous in cells and serves diverse functions related to maintenance of transmembrane Na+ and K+ gradients. Although its importance is often regarded mainly in relation to its homeostatic roles, the Na+ pump is actually a family of enzymes heterogeneously distributed in the brain and subject to regulation by many endogenous factors, suggesting the existence of a broad range of Na+ pump functions. This project seeks to test the general hypothesis that regulation of the Na+ pump provides an important mechanism for the control of neuronal excitability. Reversible, partial Na+ pump inhibition and stimulation will be used, in conjunction with electrophysiological techniques, in the rat hippocampal slice to study the mechanisms by which the Na+ pump affects excitability. The major focus is on epileptiform activity induced by partial Na pump inhibition. Specific aims of the project will test the following hypotheses: 1. PPI causes epileptiform activity by selectively reducing synaptic inhibition. 2. Ion fluxes through synaptic channels can lead to Na pump activation. 3. PPI induces a persistent increase in excitability in CA3 through an LTP-like process. 4. PPI causes an LTD-like suppression of synaptic responses in CA1. 5. PPI suppresses burst discharge processes in slices from juvenile animals. Na+ pump dysfunction has been implicated in a number of disease states, including epilepsy, but the wealth of information on the molecular biology and biochemistry of the Na+ pump is not matched by a comparable understanding of its physiological roles. This proposal is a step towards filling the gap.

This is an application for renewal of a training program in cellular and integrative neuroscience at the University of Maryland, Baltimore (UMB), School of Medicine. The goal of the program is to provide multi- disciplinary training to postdoctoral fellows based on the philosophy that neuroscientists require expertise in many facets of integrative neuroscience in order to participate effective in advanced investigations of the nervous system. Cell-cell communication is central to the integrative properties of the nervous system and plasticity of communication is increasingly recognized as a broad fundamental principle; indeed, plasticity of cell-cell communication is the organizing theme of the program. Six interactive interest groups, representing the research strengths of the program faculty, constitute focal points for investigation and training. These interest groups are: 1) neural systems, 2) cell-cell communication, 3) molecular aspects of cell-cell communication, 4) developmental neuroscience, 5) glia and 6) the application of optical methods in neuroscience. The program steering committee, an interdepartmental group of five senior investigators, is responsible for the administrative operation of the program including evaluating applicants and awarding stipends, monitoring trainee progress, coordinating recruitment efforts, and conducting an annual retreat. The steering committee and twelve additional established neuroscientists with long-standing track records of publications, successful postdoctoral training experience and extramural funding, constitute the primary faculty in whom rests the principal responsibility for supervision of trainees; several secondary faculty members collaborate with the primary faculty in research and training. In light of the explosive growth of outstanding neuroscience research at UMB, consistent success in competing for NIH funding and the large number of excellent postdoctoral fellows already trained by program faculty, this application requests support for eight fellows.

DESCRIPTION (ADAPTED FROM THE APPLICANT'S ABSTRACT): This is an application for predoctoral training support for the Program in Neuroscience at the University of Maryland, Baltimore (UMB). This Program, chartered by the State of Maryland to offer the Ph.D. in Neuroscience, offers broad-based multidisciplinary training through study tracks in four areas of neuroscience: Behavioral/Systems, Cellular/Molecular, Developmental and Cognitive/Computational. These areas reflect the research strengths of the faculty and the educational and training opportunities of the Program are focused on them. The Training Program is founded on the well-established administrative structure provided by the Program in Neuroscience and is run by an interdepartmental faculty group with individual subcommittees dedicated to advising students and monitoring their progress throughout their graduate education. General and specific course requirements, including a wide range of didactic course electives and laboratory rotations, establish an orderly, but flexible, educational framework. Journal clubs and seminars provide formal and informal educational experiences. The faculty, members of which come from the Medical, Dental and Pharmacy Schools, and the Maryland Psychiatric Research Center, is an outstanding group of 49 individuals selected for their widely recognized expertise in neuroscience research, extensive records of success in graduate training, state-of-the-art research facilities and exceptional abilities to secure national grant funding. Faculty members have numerous collaborative, teaching, and supervisory interactions, which provides cohesiveness to the program and copious opportunities for students to obtain experience in interdisciplinary neuroscience studies. The Program in Neuroscience, although established relatively recently, builds on a long and successful history of graduate training in neuroscience at LIMB. The Program, however, provides a coherence and an accessibility to interdisciplinary interactions often difficult to obtain under traditional departmental, school-based, units. The six predoctoral stipends requested each year in this application would be used to support outstanding students for the first two years of their studies prior to their embarking full-time on laboratory research in one of the interdisciplinary research tracks in the program.

DESCRIPTION (provided by applicant): Cannabinoids, the bioactive ingredients in marijuana and hashish, affect the brain by acting at specific, membrane bound. G-protein-coupled receptors (CB1s). Endogenous ligands for CB1 exist (endocannabinoids), but information on receptors, receptor localization, endogenous agonists and associated second-messenger systems does not reveal the detailed workings of the endocannabinoid system, i.e., the cells that release endocannabinoids and the cells that respond to them. At the cellular level almost nothing is known about endocannabinoid release or its functional roles. Depolarization of hippocampal CA 1 pyramidal neurons releases endocannabinoids, which bind to CB 1s on presynaptic terminals of a sub-class of interneurons synapsing on them. Activation of CB1s causes a transient decrease in GABA release. This process, called DSI (depolarization-induced suppression of inhibition), represents a unique means for detecting the release and studying the actions of endocannabinoids. Using electrophysiological and optical recording techniques on hippocampal slices from normal rats and mice, and from genetically altered mice, we will test the hypothesis that neuronal excitability is governed by the endocannabinoid system. Details of the functional roles of endocannabmoids in CAl DSI remain unclear. Moreover, endocannabinoid receptors exist in other parts of the brain, and it is not known if they perform the same functions everywhere. The dentate gyrus (DG) differs from CA 1 in many ways, including neuronal circuitry and mechanisms of plasticity, such as long-term potentiation (LTP). CB 1s in the DG are associated with the same class of interneurons as in CAl. The DG is an ideal model for testing the generality of the endocannabinoid hypothesis. The DG is a gateway to the hippocampus from extrahippocampal areas and so understanding how plasticity is controlled there will be especially important. Finally, a major scientific and clinical problem is "tolerance" to the application of exogenous cannabinoids. It is not known if tolerance to exogenous cannabinoids affects the endocannabinoid system. Our Specific Aims are to test the hypotheses: 1. That endocannabinoids are necessary and sufficient to mediate DSI. 2. That endocannabinoid release facilitates NMDAR-dependent LTP induction in CAl. 3. That mediation of DSI is a function of endocannabinoids in dentate gyrus. 4. That endocannabinoid release facilitates induction of LTP in dentate gyrus. 5. That the development of tolerance to exogenous cannabinoids affects the endocannabinoid system.

DESCRIPTION (provided by applicant): This application requests support for years 15-20 of a highly successful training program in cellular and integrative neuroscience at the University of Maryland School of Medicine (UMSOM). Our goal is to provide multidisciplinary training to highly-qualified postdoctoral fellows based on the concept that neuroscientists require expertise in many facets of integrative neuroscience in order to participate effectively in advanced investigations of the nervous system. Cellular communication is central to the integrative properties of the nervous system and plasticity of communication is a broad fundamental principle;indeed, it is the organizing theme of the program. Six interactive interest groups constitute focal points for investigation and training. They are: 1) neural systems, 2) intercellular communication, 3) molecular factors in cell signaling, 4) developmental neuroscience, 5) glia, and 6) novel optical approaches in neuroscience. In keeping with our philosophy of preparing trainees for all aspects of a career in biomedical research, this application places increased emphasis on training in non-scientific aspects of the career. With support from the UMSOM administration, formal workshops and seminars in grant writing, lab management, mentoring skills, ethics in research, etc., are available to our trainees. The program steering committee, an interdepartmental group of five senior investigators, is responsible for the administration of the program. It evaluates applicants and accepts successful trainees, monitors trainee progress, and organizes an annual retreat. The steering committee and 16 other established neuroscientists with long track records of publications, successful postdoctoral training experience and extramural funding, constitute the primary faculty, which is responsible for supervision of trainees. Eight secondary faculty members collaborate with the primary faculty in research and training. Of the 24 program graduate since 2004, 23 remain in scientific careers (22 in research) in academia, government, research institutes or industry. This includes 10 Assistant Professors (9 at research universities), 9 of whom have won significant independent federal funding (2 R01's, 4 K01s, 2 R21s, and 1 R03). In view of this record, we are requesting continued support for seven trainees.

DESCRIPTION (provided by applicant): The endogenous opioid and cannabinoid systems are powerful neurophysiological modulators of excitability in the brain, and have been linked to numerous behaviors. They are also substrates for drugs of abuse. Yet despite their unique molecular and pharmacological identities, the systems often appear to be intertwined, and drugs targeted to one system can modify responses mediated by the other system. The cellular interface between opioids and cannabinoids is not generally understood: it probably varies depending on the brain structures and behaviors involved. The mammalian hippocampus has intricate endogenous opioid and cannabinoid systems which have been studied individually. However, there is virtually no information about the ways in which they overlap or how they might interact. A potentially crucial link may be provided by the neuropeptide, cholecystokinin (CCK), which potently controls actions mediated by a major opioid receptor, the f-receptor (MOR), at both cellular and behavioral levels. The endogenous cannabinoid system in hippocampus is targeted mainly to the particular set of GABAergic interneurons that also contain CCK. In hippocampus, the brain cannabinoid receptor, CB1, is almost exclusively expressed by CCK-containing interneurons. The f-opioid receptor is localized on the neurochemically-distinct, parvalbumin-containing interneurons, that express neither CCK nor CB1. The precise and complex convergence of these three systems onto interneurons that target pyramidal cells, which are the major hippocampal output neurons, cannot be a chance occurrence. The objective of this proposal is to test the Working Hypothesis that the endogenous opioid, cannabinoid and CCK systems mutually interact via hippocampal interneurons, and thereby jointly regulate the excitability and susceptibility to short- and long-term plasticity of the pyramidal cells. The prominent roles of these systems in drug abuse and major brain disorders such as epilepsy and schizophrenia underscore the significance of the inherent unresolved questions. We will use a variety of experimental approaches in in vitro brain slices, including electrophysiological, immunocytochemical, and optical techniques, to test the major hypothesis of this project. The Specific Aims are to test the following hypotheses: 1. The interneurons that are sensitive to opioids and cannabinoids can be functionally classified by their pharmacological properties. 2. CCK affects both f-opioid and endocannabinoid-sensitive interneurons. 3. Short-term inhibitory response plasticities mediated via MORs and CB1 interact with each other and with CCK. 4. Long-term inhibitory response plasticities mediated via MORs and CB1 interact with each other and with CCK.