Michael Nitabach - US grants

potassium channel; protein tyrosine kinase; biological signal transduction; recombinant proteins; voltage gated channel; phosphorylation; Xenopus oocyte; transfection; immunoprecipitation; western blottings; monoclonal antibody; tissue /cell culture;

[unreadable] DESCRIPTION (provided by applicant): Human disorders caused by neuronal ion channel dysfunction are a major source of pain, suffering, and economic hardship. Their amelioration can be greatly facilitated by understanding the normal in vivo physiological functions served by the large number of biophysically distinct ion channels present in all animals. Our long-term goal is the development of a generally-applicable transgenic toolkit that will permit the testing of hypotheses concerning the physiological functions of particular ion channel subtypes in specific neural circuits in intact behaving animals. Our approach is based on the "tethered toxin" technology, wherein peptide ion channel blockers from venomous predators are expressed as fusion proteins tethered to the extracellular side of the plasma membrane. Preliminary studies applying this approach indicate that tethered spider toxins function as cell-autonomous ion channel blockers with their expected target selectivity when expressed in specific neuronal circuits in the brains of transgenic Drosophila melanogaster fruit flies. Preliminary studies also indicate that the venoms of Australian funnel-web spiders of the Atracinae family contain a vast diversity of uncharacterized peptide toxins expected to target a wide variety of ion channel subtypes. The proposed aims are thus directed at (1) identifying novel spider toxins with high potency against neuronal ion channels and (2) determining the molecular identities of the ion channel targets of each identified toxin. The first aim will be achieved by screening for specific behavioral effects of expressing numerous different tethered funnel-web spider toxins in behavioral control circuits in the brains of transgenic flies. The second aim will be achieved by using a combination of in vitro and in vivo electrophysiological approaches to identify the molecular target(s) of each toxin identified in the first aim. The proposed research will enable the entire Drosophila neurobiology community to begin to test hypotheses concerning the roles of particular ion channel subtypes in specific neural circuits in intact behaving animals that have been refractory to traditional approaches. Because of the extensive conservation of biophysical and physiological mechanisms of neuronal function between flies and mammals, the novel toxins we identify will not only be of tremendous use for in vivo fly neurobiology, but also as pharmacological reagents for probing the structure and function of mammalian ion channels in health and disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long term goal of this proposal is to elucidate the roles of intracellular calcium signals in circadian clock neurons. Experiments will be carried out in the fruit fly, Drosophila melanogaster, which has a functionally sophisticated and anatomically well- characterized circadian control system and is uniquely amenable to application of genetic methods that target clock neurons in the intact behaving animal. In addition to canonical transcriptional feedback mechanisms, circadian oscillation also relies upon depolarization-activated ionic membrane conductances. The proposed aims explore the hypothesis that intracellular calcium signals triggered by membrane depolarization are a core component of the cellular circadian oscillator. An engineered calcium buffer protein is specifically targeted to clock neurons in the brains of transgenic flies to disrupt cellular calcium signals in the intact living organism, followed by measurement of effects on circadian rhythms of locomotor activity, cellular accumulation of known transcription factor components of the circadian oscillator, and intracellular calcium dynamics. Preliminary studies using this approach indicate that intracellular calcium buffering in clock neurons leads to dose-dependent slowing of free-running behavioral and cellular rhythms with arrhythmicity at the highest dose. The proposed aims will identify the subcellular location of the relevant calcium signals, their temporal dynamics, the detailed effects their disruption has on cellular rhythms, and the downstream calcium-sensitive signaling pathways required for their transduction. Because of the great similarity in the genetic and cellular bases for circadian rhythmicity in flies and mammals, the information that can uniquely be obtained exploiting the genetic accessibility of Drosophila in the proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in jet lag or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration. The proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in jet lag or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration.

Pain caused by activation of pain-sensing peripheral neurons ("nociceptors") is a major source of human suffering and economic loss. Activation of nociceptors and the transmission of pain signals to the central nervous system, where they give rise to the conscious perception of pain, requires the coordinated participation of a variety of stimulus- and voltage-gated ion channels. Our long-term goal is the development of pharmaceutical agents for interfering with the functions of ion channels in mammalian nociceptors to ameliorate human pain. We will employ as a source of candidate agents a newly-discovered naturally-occurring combinatorial library of diverse peptide ion channel toxins ("atracotoxins") derived from the venom of Australian funnel-web spiders. To identify members of this library with desirable activity, we will screen them for activity against ion channels known to be important for nociceptor function. The first step in the transduction of painful stimuli-such as extreme heat or cold, noxious chemicals, mechanical injury, or inflammatory mediators-is the activation of stimulus-gated ion channels in the plasma membrane of nociceptors. Once a painful stimulus is transduced by stimulus-gated ion channels into an electrochemical signal, it must be transmitted to the CNS as an electrical signal down the axon of the nociceptor. The transmission of pain signals to the CNS requires activation of voltage-gated ion channels in the endings and axons of nociceptors. We will identify blockers of stimulus- and voltage-gated ion channels in the peripheral terminals of nociceptors by screening atracotoxins against cloned nociceptor ion channels expressed in Xenopus laevis oocytes. Once we have identified a subset of atracotoxins that are active against cloned nociceptor ion channels, we will test each of them against the corresponding native channels in acutely cultured sensory ganglion nociceptors. This secondary screen is essential for excluding from future in vivo studies of candidate analgesics those atracotoxins that are inactive against ion channels in their native state in the nociceptor plasma membrane. These studies will identify lead pharmaceutical agents for use as novel treatments for severe human pain with significant advantages over those that are currently available. In particular, by targeting ion channels present in the peripheral terminals of nociceptors, we open up the possibility for local drug application to the site of peripheral pain. Once these lead agents are identified, we will collaborate with established experts to test these agents in accepted in vivo preclinical mammalian models of pain.

DESCRIPTION (provided by applicant): Our long-term goal is the mechanistic dissection of neuropeptide signals in neural circuits in vivo. We have used a variety of genetic reagents for in vivo cell-specific manipulation of three Class B1 neuropeptides whose G protein-coupled receptors signal through cAMP in fly circadian/sleep control circuits: PDF (Pigment Dispersing Factor), DH31 (fly calcitonin homologue), and DH44 (fly corticotropin releasing factor (CRF) homologue). PDF is already known to be expressed in a subset of circadian clock neurons and to be important for regulating fly daily rhythms and sleep. Our preliminary studies now suggest distinct important roles for DH31 and DH44 in controlling daily patterns of sleep and activity. Flies adapt their bimodal crepuscular pattern of rest and activity to prevailing seasonal conditions: in the winter most activity is in the evening (to avoid the chill of night), while in the summer most activity is in the morning (to avoid the heat of day). Our preliminary studies suggest that increased autocrine activation of PDF receptors (PDFR) possessed by the PDF-secreting neurons themselves underlies this seasonal shift in the balance of activity from evening to morning. We will use various genetic tools to test the hypothesis that autocrine PDFR activation induces this plastic change in circadian network properties by modulating the daily pattern of PDF secretion itself. Recent studies implicate a particular subset of non-PDF-secreting clock cells as the required recipients of this PDF signal. Our preliminary studies indicate that many of the neurons in this subset express DH31, and that - like flies lacking PDF - mutant flies lacking DH31 exhibit severely blunted circadian morning activity. Furthermore, PDF-secreting clock neurons themselves possess DH31 receptors (DH31R), thus suggesting the hypothesis that a reciprocal feedback loop between PDF-secreting and DH31-secreting clock neurons drives morning activity. We will use various genetic tools to test this hypothesis. Our preliminary studies show that DH44-the fly homologue of CRF-is expressed at high levels in a subset of neurons in the pars intercerebralis, fly homologue of the hypothalamus. Furthermore, we have performed a preliminary behavioral genetic screen suggesting that - as for mammalian CRF - DH44 signaling to central brain neurons decreases total sleep amount and increases sleep fragmentation. Based on these preliminary findings, we will use a variety of genetic tools to identify the specific DH44 receptor-expressing neurons responsible for the regulation of sleep.

DESCRIPTION (provided by applicant): Our long-term goal is the development of tools for characterizing the functions of neuropeptides in neural circuits and non-neuronal tissues in vivo in intact behaving animals. We have developed a new technology, where neuropeptides are transgenically expressed as chimeric fusion proteins tethered to the plasma membrane via hydrophobic anchors (t-peptides). T-peptides activate their cognate G-protein coupled receptors (GPCRs) with expected specificity in t-peptide-expressing cells, but without activating their GPCRs in the membranes of neighboring cells not expressing the t-peptide. T- peptides thus provide genetically encoded tools for the cell-autonomous gain-of-function analysis of neuropeptide function in transgenic animals. The Specific Aims are designed to provide a genome-wide toolkit of genetically encoded cell-autonomous pharmacologically specific activators of neuropeptide GPCRs. Because this toolkit will be based on the UAS-GAL4 binary expression system that separates cell- and circuit- specific promoter GAL4 driver transgenes from UAS transgenes containing cDNAs that encode effectors such as t-peptides, it will be of direct utility to Drosophila biologists interested in cell-specific physiological and behavioral functions of neuropeptides. More generally, the development and validation of t-peptide libraries will also provide invaluable insights enabling the generation of tools for use in addressing mammalian neuropeptide function in vivo in health and disease, including potential clinical utility in gene therapy applications.

DESCRIPTION (provided by applicant): Neural networks in the brain control sleep and circadian rhythms, key interacting processes that regulate numerous physiological and behavioral outputs. Human disorders caused or exacerbated by impaired regulation of sleep and circadian rhythms-such as narcolepsy, genetic sleep phase disturbances, jet lag, shift work, sleep deprivation, depression, etc-are a major source of morbidity, mortality, and economic hardship. Their amelioration will be facilitated by understanding how sleep and circadian rhythm control circuits function in vivo, importantly including intercellular synaptic signaling and homeostatic plasticity. One of the key features of sleep-wake regulation is the ability to rapidly transition from one state to the other, such as to wake up upon receipt of sensory stimuli signaling danger. Current models of rapid sleep state switching in mammals involve mutually inhibitory feedback loops between sleep-promoting and wake-promoting populations of neurons to implement a bistable flip-flop. Sleep flip-flop and circadian regulatory circuits rely on both classical rapid synaptic signaling, as well as small molecule and peptide neuromodulators. Our long- term goal is mechanistic dissection of synaptic communication, neuromodulation, and their interaction in sleep and circadian control circuits of the intact animal. In pursuit of this goal w combine the cell-specific neurogenetic manipulability of the Drosophila model system with whole-cell patch-clamp and functional imaging. We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, whole-cell patch-clamp, and fluorescent imaging in vivo of intracellular Ca2+ and membrane potential to analyze the functional relationships within and between the sleep-promoting and wake-promoting neurons of the mushroom body to determine how the mushroom body controls sleep bidirectionally and whether it behaves as a bistable flip-flop.We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, and whole-cell patch-clamp in intact fly brain to determine the synaptic connections that underlie the functional network. We will also test the hypothesis that one or more of the sleep- and/or wake-promoting mushroom body neuron classes encodes homeostatic sleep drive that biases the network to one or the other state.

? DESCRIPTION (provided by applicant): Neural networks in the brain control sleep and circadian rhythms, key interacting processes that regulate numerous physiological and behavioral outputs. Human disorders caused or exacerbated by impaired regulation of sleep and circadian rhythms - such as narcolepsy, genetic sleep phase disturbances, jet lag, shift work, sleep deprivation, depression, etc. - are a major source of morbidity, mortality, and economic hardship. Their amelioration will be facilitated by understanding how sleep and circadian rhythm control circuits function in vivo, importantly including intercellular synaptic signaling and homeostatic plasticity. One of the key features of sleep-wake regulation is the ability to rapidly transition from one state to the other, such as to wake up upon receipt of sensory stimuli signaling danger. Current models of rapid sleep state switching in mammals involve mutually inhibitory feedback loops between sleep-promoting and wake-promoting populations of neurons to implement a bistable flip-flop. Sleep flip-flop and circadian regulator circuits rely on classical, rapid synaptic signaling, as well as small molecule and peptide neuromodulators. Our long-term goal is mechanistic dissection of synaptic communication, neuromodulation, and their interaction in sleep and circadian control circuits of the intact animal In pursuit of this goal we combine the cell-specific neurogenetic manipulability of the Drosophila model system with whole-cell patch-clamp and functional imaging. We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, whole-cell patch-clamp, and fluorescent imaging of intracellular Ca2+ and membrane potential to analyze the functional relationships within and between the sleep-promoting and wake-promoting neurons of the mushroom body to determine how the mushroom body controls sleep bidirectionally and whether it behaves as a bistable flip-flop. We will combine neurogenetic manipulation of classical synaptic release, optogenetic neuronal stimulation, and whole-cell patch-clamp in intact fly brain to determine the synaptic connections that underlie the functional network. We will also test the hypothesis that one or more of the sleep- and/or wake-promoting mushroom body neuron classes encodes homeostatic sleep drive that biases the network to one or the other state.

DESCRIPTION (provided by applicant): In 2006 Yale started the innovative Medical Research Scholars Program (MRSP) with the mission of providing broad and deep Ph.D. training in the core basic science concepts of molecular biology, cell biology, biochemistry, genetics, physiology, pharmacology, and pathology, integrated with intensive exposure to medically oriented coursework and mentored clinical experiences designed specifically for Ph.D. students. The ultimate goal of this training is to prepare our students to be future interdisciplinary leader in the biomedical sciences, with a unique ability to pursue clinically relevant fundamental basic science inquiry aimed at elucidating the molecular mechanisms of disease. The MRSP has been supported by a combination of institutional funds and funds provided by the Howard Hughes Medical Institute (HHMI). The combination of substantial unmet demand from highly qualified students for admission (warranting expansion of the size of MRSP), the termination of HHMI support after the 2013-2014 academic year, and the desire to continue this highly successful ongoing program constitute the rationale for our Molecular Medicine (MM) Training Grant application. MRSP students participate in a variety of program activities designed to engage Ph.D. students in medically relevant training and experiences throughout their graduate studies. The classes focus first on normal human physiology, organ-based cell biology, and biostatistics, followed by human pathobiology and an introduction to drug discovery, validation, and clinical trials. MRSP students also have the option to participate with first-year Yale MD students in weekly small-group physiology case conference tutorials, in which they explore in depth the physiological underpinnings of particular disease states. The cornerstone of the MRSP is a two-year mentored clinical experience that provides students with in-depth exposure to the science behind human diseases and first-hand longitudinal encounters with patients to contextualize the mechanistic basis of disease in a way that is not possible through traditional classroom learning. In addition, MRSP students participate in a special research-in-progress series held jointly with the Yale Center for Clinical Investigation (YCCI)-an NIH-funded center that forms the nucleus for translational and mechanistic disease-focused research at Yale-and as the program grows we plan to incorporate an Annual Retreat (also in conjunction with the YCCI). In addition to these formal activities, the MRSP also provides ongoing mentoring to all MRSP trainees regarding selection of a thesis research laboratory and assembly of the dissertation committee to support the goal of the NIGMS Molecular Medicine program to provide training in the basic biomedical sciences with a focus on elucidating the mechanisms of human disease. Accordingly, we request funds for this MM Training Grant to support a total of five MRSP Ph.D. students per class year, with five second-year and five third-year students being supported at any given time (all first-year students are fully supported by institutional funds), with the expectation that MRSP students will complete the Ph.D. within six years.

Animals learn to associate otherwise neutral sensory cues with positive or negative contingencies and rely on those associations to make adaptive decisions. Flexible updating of these acquired associations as contingencies change is also important. Failure to update internal representations plays a causal role in some mental disorders, including schizophrenia and anxiety. While the neural substrates of acquiring and updating associations have been studied in mammalian models, the complexity of the mammalian brain has made it difficult to obtain precise cellular and synaptic mechanistic understanding. Drosophila flies exhibit flexible associative learning: they learn to avoid an odor paired with electric shock, and extinguish that learned association when the odor is later presented without shock. Flies have powerful genetic tools to allow precise manipulation and visualization of neural activity with cellular resolution in the mushroom body brain region (MB), where neural plasticity underlying learning occurs. Our long-term goal is to use Drosophila to gain mechanistic insight into how acquisition and extinction are implemented by synaptic microcircuits of the MB. Our novel hypothesis is that plasticity of dopamine neurons embedded in a recurrent synaptic microcircuit residing in the fly mushroom body underlies extinction of odor-shock associations. To test this hypothesis we employ in vivo Ca2+ imaging and optogenetics to visualize and manipulate dynamic changes in neural activity of specific genetically targeted MB cell types as a fly acquires and extinguishes an association between a neutral odor and aversive electric shock.