Diane K. O'Dowd - US grants

Sensory processing and the expression of both simple and complex behaviors depend on rapid communication between disparate parts of the nervous system. This is accomplished by electrically excitable cells that encode and transmit information via electrical signals. Electrical excitability reflects the activity of voltage-gated ion channels that open in response to changes in voltage, allowing the flow of ions across the cell membrane. The properties of excitability depend on the gating behavior, number, type and spatial distribution of these channels in the membrane. changes in these properties have been implicated in modifications of excitability observed during development, following disease or injury to a nerve. A detailed knowledge of the function and regulation of ion channels at the molecular level is prerequisite to understanding the control electrical excitability in developing or pathological systems. This will require the application of electrophysiological, genetic, and molecular biological techniques to the study of voltage-gated ion channels. A powerful system in which all of these approaches can be used is Drosophila. In this proposal focus on sodium and calcium channels Drosophila neurons and myotubes and characterization of genes important in their expression through physiological analysis of mutants. I have identified three genes, para, sei, and tip-E, which alter the macroscopic sodium currents in embryonic neurons. My physiological experiments suggest that para codes for a functional sodium channel protein, consistent with its sequence similarity to a vertebrate sodium channel gene. Although sei and tip-E may also code for sodium channels, they may instead be genes which regulate other aspects of excitability such as number or distribution of channels. Further analysis of mutations at these and additional loci found to affect sodium channels, both at the whole-cell and single channel level will be important in understanding the role of these genes in sodium channel expression. In addition to sodium, calcium ions are important charge carrier in excitable cells. I therefore propose to examine calcium channels in wildtype neurons and myotubes. Genes involved in expression of calcium channels will be identified using a deficiency screen that we have developed to detect chromosomal deletions that alter the expression of whole-cell calcium currents. The results of these studies will define and characterize the molecules important in the function and regulation of ion channels involved in electrical excitability.

The thalamocortical synapse is a crucial link in sensory processing by the neocortex, and its early stages of development are particularly susceptible to abnormal sensory inputs. It may also play an important role in the propagation of epileptic seizures. Thus understanding its development is essential for understanding both normal and abnormal cortical function. Nevertheless surprisingly little is known about its properties at these early stages. The development of a novel slice preparation that retains functional thalamocortical connectivity, together with the recent application of the whole-cell recording technique to the thick slice, now make it feasible to study development of this synapse at the intracellular level. Our preliminary studies in acute thalamocortical slices have revealed that functional thalamocortical contacts are first established in the deep layers (V/VI) of the cortex and that both NMDA and nonNMDA-type receptors mediate the newly formed thalamocortical connections. The establishment of intracortical inhibitory connections is delayed relative to the formation of thalamocortical excitatory connections. In this study we propose to further examine both the morphological and physiological development of the thalamocortical synapse in neonatal mouse cortex in the acute slice. Based on electrophysiological and pharmacological criteria, responses will be classified as NMDA or non-NMDA type EPSC's or as GABAergic IPSC's. The development of each type of response will be examined separately. Properties that will be examined include the spatial distribution across different layers and on different morphological classes of neurons during the first two weeks postnatal. Labelling of the population of thalamic afferents with DiI in acute slices will be done in conjunction with the physiological experiments in order to correlate the morphological distribution of fibers with the location of functional connections. We have also determined conditions under which thalamocortical slices prepared from early postnatal animals (P0/P2) survive and maintain intact fibers projecting from the thalamus to the cortex for at least 1-2 weeks in a serum-free chemically-defined medium. In these cultures we will examine the growth of thalamic afferents and patterns of arborization in the first two weeks in culture. Electrophysiological recordings will be performed to assess which thalamic fibers projecting to the cortex form functional connections. Manipulation of the growth conditions will then be done in order to identify physiological or biochemical factors involved in the process of synaptogenesis. The results of these studies on acute and cultured thalamocortical slices will contribute to our understanding of the development of both normal and abnormal cortical function.

RESEARCH PROPOSED: The research proposed is an excellent series of experiments to first, evaluate the role of phosphorylation in regulating the function of drosophila sodium channels, and the to determine the complement of ion channels expressed by drosophila neurons by molecular fingerprinting of PCR products from single neurons. These studies complement each other well: the studies on phosphorylation and regulation of alternately spliced domains of drosophila para sodium channels are a clear extension of the ongoing research, and utilize state-of-the-art techniques with which she is familiar, such as single- cell PCR, while the last specific aim consists of experiments that utilize less explored techniques, but could provide significant new information and provide her with data that would continue her research in the area of control of ion channel expression during development.

DESCRIPTION (Verbatim from the Applicant's Abstract): Synaptic plasticity plays an important role in a number of fundamental processes in all higher organisms including development, regeneration, learning and memory. The goal of this application is to identify genes/signal transduction pathways important in regulating central synaptic plasticity, using Drosophila as a simple model system. Studies at the NMJ in Drosophila have demonstrated that a number of mutants identified on the basis of learning and memory deficits, define genes that are important in regulation of transmission and plasticity at the peripheral glutaminergic synapse. However, limited electrophysiological access to neurons in the adult fly CNS has precluded assessment of the role of these genes in regulating central synaptic transmission. This application makes use of dissociated cell cultures in which spontaneously active, excitatory cholinergic synapses form between primary embryonic Drosophila neurons, providing the first opportunity to explore the functional properties of transmission at central synapses in this organism. Electrophysiological studies will be used to identify mutations that disrupt one or more aspects of cholinergic synaptic transmission. Structural and molecular studies in mutant and wild type neurons will be important in further defining the underlying mechanisms regulating synaptic transmission during development and in differentiated neurons. The principal investigator's preliminary data demonstrates altered transmission at cholinergic synapses in dunce, a learning and memory mutant with defects in cAMP-signaling. Biochemical studies in differentiated wild type neurons have revealed that cAMP-dependent plasticity at central cholinergic synapses is likely to be important in experience-dependent memory formation in Drosophila. The use of protein/RNA synthesis inhibitors and transgenic flies to regulate dCREB2 expression are proposed to examine the mechanisms involved in cAMP-dependent plasticity at these synapses. Identification of additional genes that are important in regulation of transmission will focus on learning and memory mutants that have not been directly linked to the cAMP-signaling pathway (latheo, leonardo). The results of the studies will provide the first insights into the cellular and molecular mechanisms of central synaptic plasticity in Drosophila. The contribution of these studies to the principal investigator's general understanding of synaptic plasticity, likely to be highly conserved between vertebrates and invertebrates, will be useful in designing treatments aimed at regulating synaptic function disrupted by developmental disorders, disease, injury or during normal aging in humans.

Recent studies suggest that modifications in the number and function of nAChRs, by exposure to nicotine, plays an important role in the process of addiction. However, the molecular mechanisms important in regulating these receptors in the mammalian CNS remain poorly understood. In this grant we propose to address this question in Drosophila, a simple, genetically accessible model system. We have recently demonstrated that nAChRs, blocked by both curare and alpha-BTX, mediate the primary form of fast excitatory transmission between cultured Drosophila neurons, providing a unique opportunity to explore the genetic and biochemical signals important in regulation of these receptors and transmission at central cholinergic synapses. Our initial experiments will focus on characterization of the properties of the whole cell currents induced by acute exposure of cultured wildtype neurons to various concentration of nicotine. The second specific aim will examine the role of nicotine in regulation of the-functional nAChRs. Potential changes in the biophysical properties of these receptors will be assessed by monitoring the electrophysiological properties of nicotine-induced, ACh-induced, and cholinergic excitatory postsynaptic currents (EPSCs), following chronic exposure to nicotine. To determine if there are changes in the numbers of receptors, 125l-alphaBTX binding will be monitored before and after exposure to nicotine. In the third aim we will examine the effects of short duration, repetitive exposure to nicotine, mimicking the exposure that occurs during smoking, on nAChR expression. Finally, we will examine the properties of nicotine induced currents and regulation of nAChRs in neurons from dunce, a learning and memory mutant with defects in CAMP-signaling. Differences in the electrophysiological and pharmacological properties of the nAChRs and cholinergic synaptic currents between dunce and wildtype neurons will be important in determining if nicotine activates the same CAMP-signaling cascade that is important in reinforced learning paradigms in flies. The results of our studies will provide important insights into the molecular mechanisms underlying nicotine induced regulation of nAChRs. These data will also contribute to the general understanding of the molecular mechanisms important in regulating transmission at excitatory synapses, likely to be highly conserved between vertebrates and invertebrates, and therefore should aid in the design of drugs and/or therapies aimed at modulating plasticity in synaptic pathways contributing to addiction in humans.

DESCRIPTION (provided by applicant): Modifications in the properties of nAChRs and excitatory synaptic transmission in neurons are thought to play an important role in the process of nicotine addiction in humans. However, the molecular mechanisms underlying behavioral responses to nicotine remain poorly understood. In this grant we propose to address these questions in a genetically accessible animal model. We have recently described the properties of nAChRs that mediate fast excitatory transmission in cultured Drosophila neurons. These cultures provide a unique opportunity to explore the cellular and molecular mechanisms important in regulation of nAChRs and cholinergic transmission at central synapses. Our first aim focuses on characterizing the biophysical, pharmacological, and molecular properties of nAChRs mediating nicotine-evoked currents/calcium transients in Drosophila neurons. In addition we will determine the function of different nAChR genes by examining the consequences of knocking out or reducing the expression of specific subunit/s in the cultured neurons. This will be accomplished by examining neurona! responses to nicotine in cultures prepared from deficiencies uncovering nAChR genes or in cultures exposed to dsRNA targeted to particular nAChR subunit/s. The second aim examines the role of nicotine in regulation of nAChRs and cholinergic transmission. Neuronal responses to nicotinefACh, 1125-aBTX binding, and the properties of cholinergic transmission will be monitored in neurons chronically exposed to nicotine. We will also examine the effects of short duration, repetitive treatments with nicotine, mimicking the exposure that occurs during smoking. The cAMP signaling cascade plays a role in regulating responses to ethanol and cocaine in both flies and mammals. In our third aim we will examine the properties of nAChRs and cholinergic synaptic currents in cAMP signaling mutants to determine if the effects of nicotine are also modulated by CAMP. Finally, we will use differential display and cDNA microarrays to identify genes that are differentially regulated by nicotine. The results of our studies will provide important insights into the mo!ecular mechanisms underlying nicotine-induced regulation of nAChRs. These data will also contribute to the general understanding of the molecular mechanisms important in regulating transmission at excitatory synapses, likely to be highly conserved between vertebrates and invertebrates. Finally, this knowledge may be useful in the design of drugs ancVor therapies targeted at ameliorating or preventing nicotine addiction in humans by regulating nAChRs and synaptic function.

DESCRIPTION (provided by applicant): A major aim of modern neurobiology is to define the cellular and molecular mechanisms contributing to memory formation. The large library of behaviorally isolated Drosophila learning mutants has been useful in identification of specific genes (including dunce, rutabaga, latheo) and signal transduction cascades (cAMP-PKA- CREB) that contribute to different aspects of olfactory associative learning. It is clear from studies at the NMJ and in embryonic cultures, that many of these genes and signaling pathways can regulate neural function. However, since neurons in the brain of the tiny adult fly have been inaccessible to electrophysiological recording, it has not been possible to use this outstanding genetic resource to explore how these genes regulate function in central circuits involved in memory formation. Our preliminary studies demonstrate that we can now routinely record from identified neurons in whole brains isolated from adult flies. Robust spontaneous activity indicates circuits are functionally as well as structurally intact. We have also developed conditions that support the formation of functional synaptic connections between identified neurons in cultures prepared from late stage pupal brains. The current proposal utilizes these two preparations and takes advantage of the molecular genetic strategies feasible in Drosophila, to examine the role of cAMP signaling in regulation of activity in neuronal circuits that process information during olfactory associative learning. Experiments are focused on antennal lobe projection neurons that receive information directly from the olfactory receptor neurons in the periphery, and Kenyon cells, their targets in mushroom bodies, an association area involved in formation of olfactory memory. The results of these studies should [provide unique insights into the cellular and molecular mechanisms involved in regulation of activity in central circuits that process information during olfactory associative learning.

DESCRIPTION (provided by applicant): Influx of calcium through voltage-gated channels regulates a wide variety of cellular processes including excitability and chemical synaptic transmission in the nervous system. Calcium channels are composed of multiple subunits with the 11 subunits forming the ion conducting pores. In vertebrates there are three major families of 11-subunits genes (Cav1, 2 and 3) with multiple genes/family and some redundancy in function. Drosophila has one gene per family and these have non-redundant functions, an advantage for assessing function of distinct subtypes in vivo. The cacophony (cac) gene in Drosophila, like its Cav2 vertebrate family homologues, encodes channels responsible for regulating evoked release of neurotransmitter at the NMJ. However, the properties of Cac channels have been difficult to examine directly since electrophysiological access to the presynaptic nerve terminal is limited and the cac gene has not been successfully expressed in heterologous systems. In our recent analysis of cac mutants, using a preparation we developed to record from identified neurons in brains of adult flies, we found that Cac channels underlie PLTXII- sensitive calcium currents that regulate AP-independent release of neurotransmitter at central cholinergic synapses in the antennal lobe. A second functionally distinct calcium current is also present in antennal lobe neurons. This grant proposes three aims: 1) identify channel subtypes that underlie calcium currents in specific populations of antennal lobe and mushroom body neurons in the adult brain, 2) test the hypothesis that Cac channels regulate evoked transmission and plasticity in antennal lobe and mushroom body circuits, and 3) determine how calcium channels and G-protein signaling regulate burst firing and network activity in the antennal lobe. The results of these studies should provide insights into how specific calcium channel subtypes regulate excitability, synaptic transmission, and network activity in central circuits activated during olfactory associative learning in the adult brain. Our studies include use of cac mutant flies that exhibit temperature sensitive paralysis and seizures. Mutations in the CACNA1A gene, the human homolog of cac, are linked to neurological diseases that are characterized by a broad spectrum of symptoms including ataxias and migraines. Thus knowledge of the cellular mechanisms contributing to the behavioral deficits in cac mutant flies should also contribute to understanding of human disease associated with mutant calcium channel genes. PUBLIC HEALTH RELEVANCE: The proposed study uses Drosophila as a model organism to determine how different calcium channel subtypes regulate activity in identified circuits in the adult brain. Knowledge of the cellular mechanisms contributing to behavioral deficits in flies with mutations in voltage-gated calcium channels should contribute to understanding of human neurological diseases associated with altered calcium channel function.

Though there are many critical junctures for improving STEM education, the first two years of undergraduate education are certainly among them. Undergraduates' first experience with university STEM courses typically takes place in large lecture courses. These courses are often criticized for focusing too much on providing information and too little on fostering scientific discussion, analysis, and reflection. Many would tie perceived inadequacies of large lecture courses with the high attrition rate that takes place in STEM majors in the first two years of undergraduate study, especially among underrepresented minority students. At UCI and elsewhere, there is a great deal of interest in improving lecture courses to foster greater scientific understanding and improve retention in the STEM disciplines. Practices that are considered particularly promising for accomplishing this include enhanced faculty-student interaction; enhanced peer interaction; greater attention to problem-solving; more opportunities for personalized learning; opportunities to receive and communicate information across diverse channels and modalities; and more data-based instruction, in which faculty evaluate the effects of their own teaching by gathering and weighing evidence.

In this project a team of UCI faculty members in the School of Education and the School of Biological Sciences will carry out a systematic study of current instructional practice in large introductory STEM lecture courses at UCI. The goals are to (a) develop a comprehensive matrix for measuring instructional practices in higher education; (b) establish baseline data; (c) obtain a synoptic view of current STEM instruction; (d) promote synergy across departments and schools to conduct and share systematic evidence-based instructional research; and (e) prepare to apply for full-scale funding to support further efforts at strengthening evidence-based STEM education at UCI. The proposed study will take place among five large UCI schools that teach various disciplines within STEM: Biological Sciences, Engineering, Information and Computer Sciences, Physical Sciences, and Social Ecology. In each of these five schools, large introductory lower-division courses will be identified and six course/sections in each school will be selected for inclusion in the study. Purposeful sampling will ensure the broadest range of faculty participation, by professorial level, gender, and background. Data on current instructional practices in those classes will be gathered from three sources--live and video-taped observations of lectures, discussion sections, and lab sections; interviews with course instructors; and review of course syllabi and other materials--using rubrics that are designed to capture the relative presence or absence of the evidence-based and promising practices described above. Data analysis will focus on triangulation among these three sources to provide a broad and thorough overview of the extent to which evidence-based instructional practices are deployed in STEM lecture courses at UCI.

DESCRIPTION (provided by applicant): Mutations in the SCN1A gene encoding Nav1.1 voltage-gated sodium channels result in a variety of human seizure disorders. These include Dravet Syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Both DS and GEFS+ are autosomal dominant disorders but relatively little is known about the cellular mechanisms underlying seizure generation. Here we propose to assess the functional consequences of disease causing mutations on neuronal activity using two complementary, genetic model systems: knock-in Drosophila with SCN1A mutations and iPSC-derived neurons from patients with the same mutations. Our preliminary data demonstrate that knock-in of a GEFS+ SCN1A mutation (K1270T) into the Drosophila sodium channel gene, para, causes a semi-dominant temperature-induced seizure phenotype. Electrophysiological studies of GABAergic interneurons in the brains of adult GEFS+ flies reveal a novel cellular mechanism underlying heat-induced seizure. Consistent with disease symptoms in humans, the seizure phenotype caused by knock-in of a DS mutation (S1231R) is more severe than GEFS+. The congruence of the genotype-to-phenotype map between flies and human in this genetic disease model paves the way for use of knock-in Drosophila to study the mechanisms underlying these complex human genetic disorders. The first two aims are focused on use of Drosophila sodium channel knock-in lines to further explore the underlying cellular mechanisms contributing to heat-induced seizures and as a low cost, high efficiency, platform for discovery of genetic modifiers and drugs that suppress the seizure phenotype. In specific Aim 3 we will employ our expertise in stem cell biology to conduct parallel studies of neuronal activity in iPSC-derived neurons from patients with the same GEFS+ mutations examined in knock-in flies. Identification of common cellular mechanisms in these two model systems has the potential to identify targets for development of novel therapies to reduce or eliminate seizures in humans with epilepsy.