F Rob Jackson - US grants

In both vertebrates and invertebrates, endogenous clocks governing the expression of biological periodicity are localized in the central nervous system. One such clock in the fruitfly Drosophila has been the subject of extensive genetic and molecular studies. These studies have resulted in the identification of several genes whose products determine the properties of biological clocks in Drosophila. One of these genes, per, has been cloned and sequenced, making it possible to elucidate the structure of a clock protein. The presence of per-homologous transcripts in deletion-bearing mutants lacking the per locus suggests that a family of related clock genes exists in Drosophila. Dr. Jackson has begun to test this hypothesis by using reduced-stringency hybridization conditions to isolate per-homologous clones from phage/Drosophila cDNA and genomic libraries. In order to further characterize these putative biological rhythm genes, he proposes the following studies. (1) He will characterize the tissue and developmental expression of per-homologous genes. (2) The chromosomal location of these genes will be determined, and (3) genetic methods will be used to assess their role in the development and maintenance of biological rhythms. (4) The complete DNA sequence of one of these related genes will be derived in order to predict the primary structure of a per-homologous protein. Completion of these aims will provide information on the genetic basis of biological rhythms.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the nervous system. Alterations in GABA neurotransmission have been implicated in the etiology of several human neurological and psychiatric disorders. The synthesis of GABA is catalyzed by the enzyme glutamate decarboxylase (GAD) in vertebrates and invertebrates. Using a cloned mammalian GAD cDNA as a probe, we have isolated homologous cDNA and genomic DNA clones from Drosophila melanogaster. The clones appear to represent a single gene which is located in polytene chromosome region 64A. Limited cDNA sequencing demonstrates that the Drosophila gene encodes a GAD-homologous protein. In order to initiate developmental and molecular studies of GABA neurotransmitter metabolism, the following investigations are proposed. (1) The complete sequence of the Drosophila GAD- homologous protein will be deduced from sequence of cDNA clones. Gene dosage and protein expression techniques will be employed to verify that the Drosophila gene encodes functional GAD protein. (2) Distinct forms of GAD have been documented in mammals and insects, including Drosophila. As a prelude to studying the expression and functional significance of GAD isoforms, we will determine how many genes encode the distinct forms of enzyme. 3) The normal ontogeny and anatomical distribution of GABAergic neurons will be determined by examining the developmental and tissue expression of GAD gene RNA as well as GAD and GABA immunoreactivity. (4) Unconditional and temperature-sensitive GAD mutations will be isolated for use in genetic studies of GABAergic systems. (5) Temperature-sensitive GAD mutations will be used to eliminate GAD activity at specified developmental stages in order to assess the effects of GABA deficits on the differentiation and/or maintenance of GABAergic and other neural pathways.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the nervous system. Alterations in GABA neurotransmission have been implicated in the etiology of several human neurological and psychiatric disorders. The synthesis of GABA is catalyzed by the enzyme glutamate decarboxylase (GAD) in vertebrates and invertebrates. Using a cloned mammalian GAD cDNA as a probe, we have isolated homologous cDNA and genomic DNA clones from Drosophila melanogaster. The clones appear to represent a single gene which is located in polytene chromosome region 64A. Limited cDNA sequencing demonstrates that the Drosophila gene encodes a GAD-homologous protein. In order to initiate developmental and molecular studies of GABA neurotransmitter metabolism, the following investigations are proposed. (1) The complete sequence of the Drosophila GAD- homologous protein will be deduced from sequence of cDNA clones. Gene dosage and protein expression techniques will be employed to verify that the Drosophila gene encodes functional GAD protein. (2) Distinct forms of GAD have been documented in mammals and insects, including Drosophila. As a prelude to studying the expression and functional significance of GAD isoforms, we will determine how many genes encode the distinct forms of enzyme. 3) The normal ontogeny and anatomical distribution of GABAergic neurons will be determined by examining the developmental and tissue expression of GAD gene RNA as well as GAD and GABA immunoreactivity. (4) Unconditional and temperature-sensitive GAD mutations will be isolated for use in genetic studies of GABAergic systems. (5) Temperature-sensitive GAD mutations will be used to eliminate GAD activity at specified developmental stages in order to assess the effects of GABA deficits on the differentiation and/or maintenance of GABAergic and other neural pathways.

Mammals and invertebrates possess endogenous circadian pacemakers (clocks) within their nervous systems that regulate the expression of daily rhythms in various physiological processes including human sleep/wake states and mental performance. In the fruitfly Drosophila, a molecular genetic approach has been applied to identify and characterize the genes that determine the properties of circadian rhythms. This proposal will focus on the molecular analysis of a Drosophila gene known as miniature-dusky (m- dy). Our previous genetic analyses have shown that certain dy mutations lengthen the period of two types of circadian rhythms. Interestingly, these and other dy alleles also reduce cell size in the adult wing. Using molecular genetic techniques, we have cloned and defined the physical limits of the m-dy locus. Transcriptional activity of the locus has been assessed using RNA blot and RNase protection procedures. The latter studies have identified a transcript that is expressed in the Drosophila head, the anatomical site of the circadian pacemaker. This transcript cannot be detected in head RNA from dyn3, a mutant which has altered circadian rhythms. Moreover, the developmental expression of a different transcript is altered by a mutation known as dy73. The present application proposes molecular studies to understand the cellular function of the m-dy locus. The complete developmental pattern of gene expression will be delineated in wild-type flies and several m-dy mutants including dyn3 and dy73. P-mediated DNA transformation techniques will be used to study the relationship between the wing and circadian rhythm phenotypes of dy mutants. Sequence analyses of wild-type and mutant DNAs will be carried out to deduce the primary structure of and define functional domains within m-dy proteins. Antisera will be generated and used in immunolocalization studies to examine the pattern of protein expression within the Drosophila nervous system and other tissues. Information about the structure and distribution of this protein is essential for determining how its cellular function is related to the expression of circadian periodicity. An understanding of the cellular and molecular bases of circadian regulation in any organism has important implications for the etiology of certain human psychiatric and sleep disorders.

DESCRIPTION (Adapted from applicant's abstract): The Drosophila lark gene encodes an RRM-type RNA-binding protein that is conserved among flies, mice and humans. Our previous studies of lark indicate that it acts as a repressor protein in the circadian clock signaling (output) pathway which mediates the regulation of adult eclosion (the emergence of adults from pupal cases). Work from the lab also indicates that the clock regulation of eclosion occurs via a rhythm in lark protein abundance. We have recently shown, for example, that lark protein abundance changes during the daily cycle, with steady-state amounts being lowest several hours prior to adult eclosion. Based on our studies, we hypothesize that decreases in levels of the lark repressor lead to activation of the eclosion output pathway and the initiation of the eclosion process. This application proposes studies that will lead to a more complete understanding of lark's role in the clock regulation of adult eclosion. We propose to: (1) Further characterize the rhythm in lark protein abundance and determine whether it depends on the activity of clock proteins such as Period and Timeless; (2) Determine whether the rhythm in lark protein abundance results from diurnal changes in the translation of lark mRNA or through posttranslational alterations of protein stability; (3) Test the hypothesis that lark acts as a negative regulator of the clock output pathway mediating eclosion; (4) Pursue structure/function studies of the putative lark RNA-binding domains with the intent of defining which are relevant for the several functions of the gene; (5) Identify the in vivo mRNA targets of the lark protein. All of these experiments will address specific questions and test explicit hypotheses about the cellular and biochemical functions of lark protein. Although oscillating mRNAs and proteins have been documented in various organisms, lark represents the only functionally characterized circadian clock output component. In all organisms including humans, the clock regulation of rhythmic process must rely on comparable signaling pathways. Indeed, we have identified lark-gene homologues in mice and humans. Thus, Drosophila provides an excellent invertebrate model system for functional studies of circadian clock output pathways. In addition, the Drosophila system is also a good general model for the analysis of RNA-binding functions in an in vivo context. Finally, our proposed studies will yield reagents and experimental designs that should enhance the Drosophila system as a model for the study of other RNA-binding functions.

DESCRIPTION: (Applicant's Description) Conference Goals: The Chemotherapy Gordon Conference is designed to promote discussion, collaboration, and progress among scientists interested in the chemotherapeutic mannagement of cancer; investigators at the basic and clinical levels are included. In addition to reviewing the latest ideas, new drugs, and experimental treatments in cancer chemotherapy, an attempt is made to review new concepts in basic tumor biology that may yield new therapeutic targets. One of the primary goals of the meeting is to promote translational research by bringing together people involved in clinical and preclinical drug development with basic scientists working on potential new targets; one of the most important questions being addressed is: "how can the discoveries of the last decade in cancer biology and genetics be applied to improving cancer treatment?" Out meeting thus differs from many other cancer research meetings in that the primary focus is on turning new discoveries into new treatment approaches. In contrast with the meetings of the last two years, which focussed on molecular and cell biology topics relevant to chemotherapy, the emphasis this year will be on new targets for drug discovery, on new drug discovery technologies, and on new agents currently in preclinical and clinical development.

DESCRIPTION (provided by applicant): The circadian system regulates a multitude of essential rhythmic physiological processes in organisms ranging from cyanobacteria to humans. Studies during the past 10 years have demonstrated a remarkable conservation of the cellular mechanisms governing circadian periodicity in insects and mammals. This renewal application proposes studies of two Drosophila RNA-binding proteins, LARK and dFMRP, that have a role in the circadian control of behavior. Preliminary studies have identified potential target RNAs for the LARK and dFMRP proteins and certain proposed experiments will use molecular and genetic strategies to determine which targets are relevant, in vivo, for circadian functions. Most LARK target RNAs contain a novel A-rich consensus sequence element in the 3'UTR, and other experiments will test the hypothesis that LARK binds to this element to regulate the stability of targets. Such a function would provide a useful mechanism for the dynamic, clock-regulated control of RNAs encoding factors important for circadian control. It is hypothesized that LARK functions in a circadian output pathway, and genetic approaches will be employed to identify additional elements of this pathway that function with the RNA-binding protein to regulate rhythmic behaviors. The cellular requirements for LARK and dFMRP, with regard to circadian functions, have not been elucidated. Therefore, in a final set of proposed experiments, cell type-specific expression methods, together with RNA interference techniques, will be employed to perturb function and determine the neuronal sites of action for these RNA-binding proteins. Altogether, these proposed studies will contribute to a better understanding of how circadian clocks control organismal physiology. These proposed studies of circadian control mechanisms have considerable significance for an understanding of normal human physiology and for the etiology of pathophysiological conditions, such as jetlag and certain sleep/wake disorders that result from environmental or genetic perturbations of the circadian system. As one of the premier eukaryotic genetic models, Drosophila is an excellent experimental organism for conducting molecular genetic investigations of the circadian system. It is anticipated that the results of studies in Drosophila will provide important and general insights about the molecular mechanisms mediating circadian control.

Conventional laser scanning confocal microscopy (LSCM) and multi- photon laser scanning microscopy (MPLSM) are now state-of-the-art technologies for vital imaging and/or the acquisition of high-resolution optical sections from thick biological specimens. This application requests funds for a Leica TCS SP2 confocal microscope to support research in Neuroscience and other areas of biomedical science at Tufts University. The Neuroscience Program of Tufts includes a strong, interdisciplinary, well-established, and well-funded group of investigators on the Boston and Medford campuses of the University. A core group of 10 investigators, all with external funding, will be primary users of the Leica TCS SP2 facility, and will account for approximately 60% of microscope use. These primary users are all faculty of the Tufts Graduate Neuroscience Program, which is intensely interactive and collaborative in nature. We will also ensure that the Leica TCS SP2 instrument is available to the larger community of faculty of Tufts Medical School; currently there are limited confocal facilities available to these faculty. The Leica instrument will be housed in a new Neuroscience Imaging Facility, with the support of the Chair of Neuroscience and the Dean of the Medical School. This facility will be supervised by a dedicated half-time technician who will have primary responsibility for maintaining the instrument and training new users. An advisory committee consisting of Neuroscience faculty has been organized to oversee the use and maintenance of the Leica instrument. Policies for the use and equitable sharing of the Leica confocal and a financial plan for the long-term maintenance of the instrument have been established by the advisory committee. At present, there is only a single, heavily-used (and outdated) confocal microscope for all three campuses of Tufts University (located within the Anatomy Department of the Medical School). Thus, the presence of the new Leica instrument and the Neuroscience Imaging Facility will greatly enhance the research of Tufts University faculty.

DESCRIPTION (provided by applicant): Understanding the cellular and molecular bases of behavior is a cardinal goal of contemporary neuroscience research, and in this regard, the fruit fly Drosophila has proven to be an excellent model system for molecular genetic investigations of behavior and the nervous system. This application proposes studies of a Drosophila circadian mutant known as Andante. This mutant exhibits a circadian long-period phenotype (about 2h longer than the wild type), and such a phenotype is observed for adult locomotor activity and adult eclosion rhythms, consistent with an effect on the molecular oscillator. In addition, a genetic mosaic analysis of Andante indicates a functional requirement within head (and presumably neural) tissues. The Andante mutation has been mapped to a small genomic interval within region 10E of the X chromosome. Recently, we identified new transposon-insertion alleles of the gene, and this has enabled us to begin to define the physical limits of the Andante transcription unit. Our preliminary studies strongly suggest that Andante encodes a Casein Kinase II (CKII) beta ortholog, a known regulatory subunit of the CKIIalpha/beta holoenzyme. Given the known importance of post-translational regulation for clock function, we hypothesize that Andante product (CKIIbeta) acts through the CKIIalpha subunit to regulate phosphorylation (and activity or stability) of one or more elements of the molecular oscillator. In this application, we propose to: (1) define the molecular limits and verify the identity of the Andante gene; (2) explore the molecular basis of the Andante phenotype by characterizing an existing Andante point mutant; (3) determine which tissues and cell types require Andante function for normal circadian periodicity; (4) define the biochemical mechanism through which Andante product (i.e., CKIIbeta) regulates clock function; and (5) directly assess the circadian requirement for CKIIalpha activity by creating and behaviorally characterizing alpha subunit variants. Because the clock mechanism is fundamentally conserved between flies and mammals, our proposed studies in Drosophila have obvious ramifications for an understanding of the human circadian clock and the treatment of human pathophysiological states that arise from alterations of circadian timing.

DESCRIPTION (provided by applicant): This application requests funds to establish a NINDS Center Core to augment the research capabilities of NINDS and other neuroscience investigators at Tufts School of Medicine (TUSM) and the affiliated medical center Tufts-New England Medical Center (T-NEMC). Within the research labs of TUSM and T-NEMC, there are 17 NINDS-funded research projects, spanning most areas of contemporary neuroscience research. Most of our Tufts NINDS investigators are members of the Sackler School Graduate Neuroscience Training Program, a highly collaborative group of 28 neuroscientists from TUSM and T-NEMC. This is a rapidly evolving program that includes 6 new neuroscience departmental faculty and a total of 12 new TUSM or T-NEMC neuroscientists hired within the last 6 years. Due to the rapid growth of our program, we have become concerned with providing adequate research core facilities to neuroscience investigators, and have held planning meetings to determine the need for additional or expanded core facilities. The award of NINDS Center Core funds will permit us to integrate faculty and research facilities from TUSM and T-NEMC, with the primary intent of providing needed services to NINDS investigators. A related goal is to foster collaborative research among the large collection of NINDS investigators at TUSM and T-NEMC. Importantly, the TUSM/T-NEMC NINDS cores will be available to other investigators at TUSM, T-NEMC, and Tufts University. The establishment of these cores is greatly aided by the expressed support of Tufts University, TUSM, and T-NEMC, who are providing assistance, funds and space for the establishment of a NINDS Center Core. The administration of the new center core will be based in the TUSM Department of Neuroscience. Supervision for the center core will be provided by the P.I. Dr. F. Rob Jackson and an advisory committee including core directors and outside scientists with relevant expertise.

This Core takes advantage of existing successful EM and Confocal Imaging Cores and will be directed by the PI (Dr. Jackson) and the present co-Directors of the existing Confocal Facility (Drs. Hunter, Jacob, and Kauer). In addition, an expert outside consultant, Dr. Kent Keyser (UAB) will also aid in the oversight and maintenance of this Core. Funds have been recently obtained that will add multiphoton capability to the Confocal Imaging Core. The multiphoton capability will greatly increase the research capabilities and progress of the Neuroscience Faculty. The new Core will also make FRET based approaches accessible to the Neuroscience Investigators. A principal goal of this Core is to provide ongoing support for personnel and equipment upgrades to allow for non-prohibitive user fees. The current scheme for user fees predicts that the hourly rate for the Confocal Facility would increase to at least $65 per hour in the absence of the requested support. Further, the addition of 2-photon capability will place increased demand on the services of the current manager, Mr. McConnell. Thus, additional technical support personnel are required to take full advantage of existing capabilities. Additional space will be provided by the administration within new Neuroscience space for one microscope. Another microscope and the EM Facility will remain where they currently are as this space is adequate.

F. Rob Jackson, Ph.D. (Proposal 0234724) "Circadian control of locomotor activity"

In all animal species, including humans, many different biological processes (e.g., sleep or general activity) show 24-h rhythms and are controlled by an endogenous circadian clock, located in the brain. In the present proposal the fruit fly Drosophila melanogaster is being used as a genetic model to study the factors that act downstream of the clock cells (in the brain) to effect the expression of rhythmic processes. One of those factors is called Ebony and is the subject of this application. The ebony gene is expressed according to a circadian rhythm and ebony mutants show altered activity rhythms. The purpose of the present work is to determine where (in which cells) the ebony gene acts in the nervous system and to study how those cells control activity rhythms. The advantage of this research approach in Drosophila is the use of genetic techniques to examine the cell biology and biochemistry underlying complex behavioral processes. As many of the factors comprising circadian clock systems are conserved between Drosophila and mammalian species (e.g., humans), the proposed work may help elucidate the clock control of rhythmic processes in other species.

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This is a supplemental application requesting funds to add instrumentation and personnel to an existing NINDS-funded center core (the Tufts Center for Neuroscience Research, CNR). Several qualifying center members and other NINDS-funded investigators at Tufts University School of Medicine and Tufts-New England Medical Center (Tufts-NEMC) have a need to employ high-density Affymetrix gene arrays in expression profiling studies. Although an expression array core exists at Tufts (the Tufts-NEMC Expression Array Core, TEAC), this facility only includes equipment for generating and analyzing cDNA or oligonucleotide gene arrays; it does not provide instrumentation for Affymetrix chip hybridization, washing and scanning. Indeed, there is not a single Affymetrix system within any department of Tufts University or Tufts-NEMC. We provide preliminary data and justifications for supplemental funds to purchase a complete Affymetrix system. The new Affymetrix system will be located within the existing TEAC facility; it will dovetail well with an existing bioinformatics core that is currently funded by our center grant. Our bioinformatics core currently provides microarray data analysis services to center members and other investigators who perform gene-profiling studies. In addition to instrumentation, this application requests salary support for a technician to maintain the new Affymetrix system and conduct experiments for NINDS and other investigators. The technician will be supervised by Dr. Dale Hunter, who serves as director of both the bioinformatics and the TEAC core facilities, as well as Ms. Lan Wei, the manager of the TEAC facility. The introduction of an Affymetrix system at Tufts University will complement existing expression array facilities and provide significant added value to the research programs of NINDS-funded investigators and other scientists at Tufts University and Tufts-NEMC. [unreadable] [unreadable] [unreadable] The written critiques of individual reviewers are provided in essentially unedited form in the "CRITIQUE" sections below. Please note that these critiques were prepared prior to the review meeting and may not have been updated or revised subsequent to any discussions at the review meeting. The "RESUME AND SUMMARY OF DISCUSSION" section summarizes the final opinion of the committee. [unreadable] [unreadable] [unreadable]

This application requests continuing support for the Tufts Center for Neuroscience Research (CNR), a NINDS-funded center that provides research core services for the Tufts neuroscience community. Our community consists of 40 neuroscience research laboratories in 14 Departments of Tufts University and its affiliated hospitals. Interests among these faculty span a wide range of medically-relevant neuroscience research areas, including synapse biology, ion channel function, sensory & behavioral neurobiology, neural signaling, neural plasticity, and neurological disease. Presently, there are 9 NINDS-funded projects among Tufts neuroscientists and a total of 44 NIH grants (> $9.5 Million in direct costs this year). The majority of these neuroscience projects depend heavily on core services that are provided by the NINDS-funded CNR. The CNR Cores have had significant impact on neuroscience research at Tufts. More than half of the Tufts neuroscience research labs use CNR core facilities, and research conducted in the cores during the last 3.5 years has resulted in at least 90 publications, many of them describing collaborative studies by Tufts investigators. The following four CNR cores are now wholly or partially supported by NINDS funds: (1) Imaging & Cell Analysis (fluorescence microscopy, confocal & 2-photon microscopy, laser capture microdissection, electron microscopy), (2) Computational Genomics (gene microarrays, Q-PCR, bioinformatics and computational biology services), (3) Animal Behavior (rodent behavior testing services), and (4) Electrophvsiology & Biophysics (single cell recording, tissue-slice field recording and tissue-slice single-cell recording). The primary continuing goal of our cores is to provide essential research services to NINDS investigators, other neuroscientists and other investigators of the Tufts community. Secondarily, the CNR continues to try to foster collaborative research enterprises among Tufts neuroscientists and to provide training and educational experiences that benefit the entire Tufts neuroscience community. Neuroscience is a major component of the recently completed Tufts Medical School strategic plan and thus we are poised to recruit new neuroscientists in the near future. During the coming expansion of neuroscience at the University, we believe that the affordable research services provided by CNR cores will greatly augment the start-up packages provided to newly-hired Tufts neuroscientists and speed their integration into the local neuroscience community. In recognition of the essential role that the CNR plays in the neuroscience research programs at Tufts, the University has provided significant institutional support to help establish the Core Facilities over this initial period¿ amounting to nearly $1,000,000 in funds and renovated space Importantly, such support will continue during the next grant cycle (see Section B3) and greatly facilitate the effort to provide core research services to Tufts neuroscientists.

This application requests continuing support for the Tufts Center for Neuroscience Research (CNR), a NINDS-funded center that provides research core services for the Tufts neuroscience community. Our community consists of 40 neuroscience research laboratories in 14 Departments of Tufts University and its affiliated hospitals. Interests among these faculty span a wide range of medically-relevant neuroscience research areas, including synapse biology, ion channel function, sensory &behavioral neurobiology, neural signaling, neural plasticity, and neurological disease. Presently, there are 9 NINDS-funded projects among Tufts neuroscientists and a total of 44 NIH grants (>$9.5 Million in direct costs this year). The majority of these neuroscience projects depend heavily on core services that are provided by the NINDS-funded CNR. The CNR Cores have had significant impact on neuroscience research at Tufts. More than half of the Tufts neuroscience research labs use CNR core facilities, and research conducted in the cores during the last 3.5 years has resulted in at least 90 publications, many of them describing collaborative studies by Tufts investigators. The following four CNR cores are now wholly or partially supported by NINDS funds: (1) Imaging &Cell Analysis (fluorescence microscopy, confocal &2-photon microscopy, laser capture microdissection, electron microscopy), (2) Computational Genomics (gene microarrays, Q-PCR, bioinformatics and computational biology services), (3) Animal Behavior (rodent behavior testing services), and (4) Electrophysiology &Biophysics (single cell recording, tissue-slice field recording and tissue-slice single-cell recording). The primary continuing goal of our cores is to provide essential research services to NINDS investigators, other neuroscientists and other investigators of the Tufts community. Secondarily, the CNR continues to try to foster collaborative research enterprises among Tufts neuroscientists and to provide training and educational experiences that benefit the entire Tufts neuroscience community. Neuroscience is a major component of the recently completed Tufts Medical School strategic plan and thus we are poised to recruit new neuroscientists in the near future. During the coming expansion of neuroscience at the University, we believe that the affordable research services provided by CNR cores will greatly augment the start-up packages provided to newly-hired Tufts neuroscientists and speed their integration into the local neuroscience community. In recognition of the essential role that the CNR plays in the neuroscience research programs at Tufts, the University has provided significant institutional support to help establish the Core Facilities over this initial period- amounting to nearly $1,000,000 in funds and renovated space Importantly, such support will continue during the next grant cycle (see Section B3) and greatly facilitate the effort to provide core research services to Tufts neuroscientists.

DESCRIPTION (provided by applicant): The human brain contains more than 100 billion cells, the majority being non-excitable glial cells. Recent studies, including those from the applicant's lab, demonstrate that glial cells of vertebrate and invertebrate nervous systems have remarkably dynamic roles in the regulation of physiological and behavioral processes. Studies in mammals have demonstrated that neurons and glia communicate with one another and this has given rise to a model of the tripartite synapse wherein a glial cell (an astrocyte) cooperates with presynaptic and postsynaptic neuronal elements to regulate communication events and behavioral processes (see Significance). Recent studies from the applicant's lab describe a role for a defined population of Drosophila astrocytes in the regulation of circadian behavior. Other studies have documented additional functions for fly glia in the regulation of neurotransmission and behavior (reviewed in Jackson and Haydon, 2008). In the present application, we propose experiments to elucidate the functions of glia in circadian timing. Our studies will employ Drosophila so as to be able to utilize sophisticated genetic techniques to study neuron-glia interactions in the circadian system. The work will utilize innovative genetic, behavioral, imaging and electrophysiological approaches and, importantly, the PI and co-I have complementary strengths in these areas. We propose three specific aims that will test explicit hypotheses about neuron-glia interactions in the circadian system: (1) Test the hypothesis that gliotransmission or other glial processes are essential for circadian behavior; (2) Test the hypothesis that glia regulate pacemaker neurons; and (3) Test the hypothesis that clock neurons regulate glial rhythms. We expect that the results of these studies will highlight general mechanisms by which neurons and glia cooperate to influence circadian rhythmicity and other behaviors. In most neurological disorders and psychiatric states, glial cell gene expression profiles are altered, and it is likely that this initiates dramatic structural/functional changes in the brain that lead to these disorders. Alterations of glial cell biology have been implicated in mental and neurodegenerative diseases including multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), schizophrenia, epilepsy, and Alzheimer's. Our proposed studies of glia and circadian control mechanisms have considerable significance for an understanding of pathophysiological conditions such as jetlag and sleep/wake disorders resulting from environmental or genetic perturbations of the circadian system. Molecular components of the circadian system are conserved between insects and mammals, including humans, and Drosophila is an outstanding model for conducting genetic investigations of circadian behavior. It is anticipated that the results of our proposed studies will provide important and general insights about the interaction of glia with the neuronal circuitry controlling behavior, insights which are critical for understanding the roles of glial cells in health and disease. PUBLIC HEALTH RELEVANCE: The human brain contains more than 100 billion cells, the majority being non-excitable glial cells; we propose studies that will utilize behavioral measures, imaging technology and other neurobiological methods to understand communication between neurons and glia of the adult nervous system. The model we propose to use for understanding neuron-glia communication is the circadian clock system as much is known about the neural circuitry responsible for circadian behavior. Our proposed studies have significance for understanding the roles of glia and neuron-glia communication in health and many different neurological diseases.

DESCRIPTION (provided by applicant): There is controversy about the importance of glia-to-neuron communication in the adult brain, notwithstanding studies of gliotransmission 1, 2 and indications that astrocytes can modulate neuronal excitability and behavior in mammalian models 3, 4. For example, some have questioned the importance of glial Ca2+-dependent mechanisms in neuronal modulation 5-7. In addition, there is still limited knowledge of the mechanisms regulating gliotransmitter release, although certain studies suggest it involves vesicular exocytosis 2. In this application, we propose unbiased molecular genetic approaches to identify glial and neuronal factors essential for glia-to-neuron communication. We previously showed that elimination of a Drosophila glial-specific factor called Ebony genetically suppressed the hyperactivity phenotype of a Dopamine Transporter (DAT) mutant 8, indicating a role for glia-to-neuron communication in the regulation of activity level. In more recent published studies, we have demonstrated that adult Drosophila glial cells can physiologically modulate locomotor activity and circadian rhythmicity9. Our studies also demonstrate a critical role for Ca2+- dependent mechanisms and Drosophila astrocyte-like glia in the regulation of locomotor activity 9. In this R21 application, we propose studies to identify cell signaling mechanisms and intracellular pathways that are important for glia-to-neuron communication in adult animals. Drosophila is an excellent model for such studies as the glial classes of the adult brain have been well characterized 10-12 and one class has developmental, morphological and molecular similarities to mammalian astrocytes 11, 13, 14. Our studies will take advantage of multiple conditional perturbation methods that are cell type-specific and reversible to identify novel, conserved factors and intracellular pathways that mediate the glial modulation of neurons and behavior. Aim 1 will utilize Drosophila strains we recently developed and translational profiling methods to identify neuronal factors and molecular pathways that are modulated by glial signaling. Aim2 will use behavioral genetic strategies to define glial signaling mechanisms that are important for neuronal modulation. This proposal represents the first broad study of neuronal proteomic changes that occur as a consequence of glial signaling. It also represents the first genetic analysis of glial mechanisms that mediate the modulation of adult neurons. As such, the results will have a major impact on glial biology - they will define molecular pathways within neurons and glial cells that mediate glia-to-neuron signaling, and set the stage for studies of conserved factors in mammalian models to understand their functions in human health and neurological disease.

DESCRIPTION (provided by applicant): Astrocytes are now recognized as active components of mature synapses; they structurally ensheath synapses and modulate neurotransmission in the central nervous system (CNS). Astrocyte dysfunction has been implicated in various neurological disorders and has been shown to actively modulate disease progression. Although astrocytes undergo a developmental maturation process in which subtypes form unique and elaborate morphologies and express overlapping but distinct molecular signatures, it is unknown how astrocyte heterogeneity arises during development of the CNS and how astrocyte development is regulated, in part because of a lack of appropriate tools for such studies. We propose to use integrated molecular and genetic approaches in Drosophila and mouse to define factors that distinguish astrocyte subtypes and regulate their developmental maturation. In particular, this project will focus on these aims: 1) Molecularly define astrocyte subtypes within the cortex and in different CNS regions using FACS and TRAP approaches; 2) Perform dEAAT1-based genetic screens to identify regulators of astrocyte development; 3) Develop new cre recombinase mice for studying astrocyte heterogeneity and function in vivo. We have generated a large amount of preliminary data demonstrating feasibility for the three aims summarized above. By characterizing molecular signatures of astrocyte subtypes in the CNS and identifying regulators of astrocyte development, this project will provide markers for astrocyte subtypes in the cortex and novel insights about how astrocyte maturation occurs. The development of a new cre recombinase driver mouse line will facilitate the selective deletion/activation of genes in astrocytes of the cortex, a region for which an existing astrocyte cre driver line is not very effective. Knowledge of astrocyte heterogeneity and new tools for studying it are critical for understanding how astrocytes become dysfunctional and how distinct classes of astrocytes contribute to the pathogenesis of psychiatric disorders.

Summary and Relevance: Genome-wide approaches involving NextGen sequencing efforts are becoming standard in neuroscience and other disciplines. As a consequence, infrastructure to support such efforts is critical for contemporary neuroscience research. During the first two cycles of NINDS funding, the CNR Computational Genomics Core (CGC) has assembled personnel, lab space, equipment, and software for qRT-PCR and the analysis of gene expression using microarray-based methods. In the next funding cycle, the CGC will continue to provide support for these efforts. In addition, the core will expand computational approaches by completing a standardized pipeline for NextGen (RNA-seq)-based approaches. Development of hardware and software to support such computational analyses will be undertaken as a joint venture with Tufts University Information Technology (UIT; see Section C5.3d). To further support RNA-seq methodology for neuroscientists, the CGC will introduce a new service for the generation of NextGen sequencing libraries. Support for NextGen sequencing, but not library preparation, already exists within another Tufts core (the Tufts University Core Facility; TUCF). Notably, the CGC remains one of the few shared resources within Tufts University and Tufts Medical Center supporting genomics approaches. Thus, the CGC is critical for evolving genome-wide analyses within Tufts Neuroscience as well as other university programs. The core has been used by 28 Tufts neuroscientists including 6 NINDS-funded investigators during this funding cycle (see later sections).

Summary and Relevance. Studies of animal behavior are necessary to characterize cardinal symptoms of disorders for which altered genetic expression and neurochemistry have been proposed. Comprehensive behavioral phenotyping, however, requires expertise and instrumentation beyond the financial and technical capabilities of most individual investigators. The mission of the ABC is to provide access to a state of the art behavioral assessment facility and expert services for assessing the behavioral effects of neuropharmacological or molecular genetic manipulations in order to identify therapeutic targets for treating CNS disorders. Since its inception in 2004, the ABC has served over 38 investigators within the neuroscience community. The ABC is a high impact resource; experiments accomplished using the ABC and its services have resulted in 13 publications and 29 NIH-funded grants. The ABC is emerging as a regional resource, serving neuroscientists within the Tufts community and drawing investigators from neighboring institutions.

The development of advanced microscopy techniques is a driving force behind advances in biological research and medical imaging. The refinement and commercialization of laser scanning confocal microscopes, nanoscopes and other types of imaging instruments along with advances in fluorophore development have led to a revolution in biological microscopy (Buckers et al., 2011;Wang et al.,2011;Reck-Peterson et al., 2010). The newest imaging methods, however, often require expertise and instruments beyond the financial and technical capacities of most individual scientists. Thus, shared imaging facilities are crucial components of university research cores. The ICAC is the only shared imaging facility within Tufts Medical School and Tufts Medical center. It was developed in response to the rapid ad vances occurring in contemporary biological microscopy and to meet the imaging priorities of Tufts neuroscience investigators. The ICAC has provided Tufts neuroscientists with a broad range of microscopic imaging services including wide-field fluorescence microscopy & digital imaging, confocal microscopy, 2-photon microscopy, Total Internal Reflection Fluorescence (TIRF) microscopy, electron microscopy (EM), live-cell imaging, and laser capture microdissection. Equipment is available for standard microscopy as well as dynamic imaging such as fluorescence resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP) experiments. The ICAC has been heavily used since its inception; it was used by 39-46 labs and 62-70 individuals during every year of this funding cycle. Thirty one neuroscience labs including 9 NINDS-funded labs employed ICAC facilities during this cycle. Given the heavy core usage, we recently added a second confocal microscope and a TIRF microscope to the facility using supplemental funds from NINDS and a shared instrumentation grant from NCRR (see Section B5

Electrophysiological data are often considered the gold standard for demonstrating a causal relationship between cell/circuit physiology and behavior. Generating preliminary data is a daunting prospect, however, for an investigator who lacks the years of training necessary to produce a competent, independent electrophysiologist. Electrophysiological studies also require a considerable up-front investment in personnel and equipment, further discouraging investigators from entering the electrophysiological arena. When the EBC was first established, the electrophysiology community at Tufts was small (Cox and Dunlap being the only electrophysiologists on the faculty), yet there were significant numbers of faculty seeking to bring electrophysiological approaches to bear on their research projects. The EBC was, thus, established to promote more widespread use of electrophysiology in neuroscience research laboratories at Tufts. This mission has been realized more completely than we thought possible (see C5.2.3i for details). Tufts' electrophysiology community has changed dramatically since the CNR was established in 2003, in part due to the success of the EBC. Several investigators received their introduction to electrophysiology in the EBC. Remarkably, four of these (Feig, Galper, Jacob, Rios) now have students and/or fellows in their labs who independently perform such experiments on setups in their own labs or in the EBC. In addition, the recent Neuroscience Department expansion has further changed the demographics of CNR users. Electrophysiological recording is now key in the labs of Cox, Dunlap, Dulla, Feig, Galper, Haydon, Jacob, Maguire, Moss, Rios, and Trimmer. With this growth of the electrophysiology community, (i) the number of users needing access to EBC's existing setups is approaching a steady-state, (ii) the majority of EBC users are sufficiently trained to not only carry out their experiments independently, but to help train future lab personnel, and (iii) there is increasing demand for in vivo stimulation/recording as programs adopt a more behavioral focus. This evolution is driving changes in the EBC (described below) to allow us to continue to meet the electrophysiological needs of the Tufts neuroscience community.

? DESCRIPTION (provided by applicant): In mammals and insects, intercellular signaling by peptides and protein hormones is critical for the functions of circuits regulating sleep and circadian behavior. In this application, we propose experiments to test the hypothesis that neuronal secretion and action of a novel immunoglobulin (Ig)-domain protein is required for proper function of the Drosophila circadian and sleep circuits. Ig superfamily molecules are important mediators of neural development, synapse formation, plasticity and injury (1;2), although less is known about the adult functions for these proteins. In recent expression profiling studies, we identified numerous Drosophila Ig protein-encoding genes that are expressed in the adult brain. Sixteen of them, including the CG141441 (Nkt) gene, have high adult nervous system expression and encode Ig proteins that are predicted to be secreted molecules. (FlyAtlas; (3)). In unpublished studies, we have shown that flies with RNAi-generated pan-neuronal deficits for Nkt exhibit reduced nighttime sleep and lengthened circadian period. Selective knockdown of Nkt in clock neurons causes a lengthened circadian period with no effect on sleep. These results suggest that Nkt is required in different neuronal cell types for th regulation of sleep and circadian behavior. Interestingly, genetic interaction with a period (pers) mutation suggests that NKT acts on pacemaker neurons and through the molecular clock to regulate circadian period. As NKT is predicted to be a small secreted Ig protein, we hypothesize that it serves a signaling function, similar to PDF neuropeptide, in the circadian circuit. In this R21 application, we propose to study the functions of NKT in the regulation of adult rhythmic behavior and sleep. Aim 1 will use conditional expression approaches to verify that NKT protein has a physiological requirement in the adult brain for the regulation of behavior. Additionally, it will delineate the neuronal requirements for the protein in sleep and circadian behavior. Aim 2 will examine genetic interactions with clock and sleep-altering mutations to better understand NKT's mechanistic functions in the relevant neuronal circuits. This aim will also ask whether NKT is secreted from neurons and test the hypothesis that the Ig protein is required for molecular oscillator function or synchronization of the clock neuron population. Proteins homologous to NKT, with single immunoglobulin domains are present in many species including C. elegans, Drosophila and humans (e.g., small secreted Neuregulin-2 isoforms). Thus, the research is likely to provide novel insights about signaling mechanisms that function in the differentiated brain.

Abstract Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. Both in mammalian and invertebrate models, there are known requirements for glial cells, in addition to neuronal circuits, in the regulation of circadian behavior and sleep (reviewed in [1;2]). Using the Drosophila model, our lab was the first to demonstrate that astrocytes were required, in vivo, for normal rhythmic behavior [3;4], and recent studies from other labs demonstrate a conserved role for astrocyte-neuron communication in the circadian systems of mammals [5;6]. Using Translating Ribosome Affinity Purification (TRAP) methods, combined with RNA-seq, we delineated the genome-wide expression patterns of Drosophila astrocytes. Based on this knowledge, we performed genetic screens to identify glial factors that regulate rhythmic behaviors. Those screens identified glial vesicle release components, an SLC-type transporter and several small secreted proteins that are required for normal circadian activity rhythms or sleep [7]. One glial gene encodes an Ig-domain protein with a high-quality signal sequence, indicative of secretion; it is required in astrocytes for the regulation of night sleep. Although astrocytes are often described as a single class of cells, accumulating evidence indicates that fly and mammalian astrocytes as well as other classes of glia are heterogeneous with many different morphological and functional subtypes [8-10]. This application proposes molecular genetic studies to identify glial subpopulations that mediate regulation of circadian behavior or sleep. Aim 1 will employ two alternative genetic methods to define glial cell subpopulations that contribute to rhythmic behavior. Aim 2 will use TRAP-seq techniques to derive the genome-wide expression pattern of glial subpopulations that are relevant for behavior with the goal of defining molecular markers of functional glial cell heterogeneity. Preliminary results documenting feasibility for both aims are included in the proposal. Together, the two aims will spatially localize glial cell subpopulations that regulate rhythmic behaviors and provide molecular markers for the further analysis of these cell populations.

Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. Both in mammalian and invertebrate models, there are known requirements for glial cells, in addition to neuronal circuits, in the regulation of circadian behavior and sleep 1-4. Using the Drosophila model, our lab was the first to demonstrate that astrocytes were required, in vivo, for normal circadian behavior 5, 6, and we show in this proposal that astrocytes also modulate sleep. In preliminary studies for this application, we have demonstrated that activation of fly astrocytes, by increasing intracellular Ca2+, promotes sleep whereas inhibition of astrocyte vesicle trafficking and release leads to decreased sleep. In related studies (now published), we have shown that an astrocyte-enriched, secreted protein known as NKT promotes sleep 7. Based on these findings, we hypothesize that neuronal sleep-regulating circuits are subject to astrocyte modulation. Aim 1a will test this hypothesis by simultaneously activating or inhibiting astrocytes and recording the excitability of known sleep- regulating neuronal populations using genetically encoded voltage sensors. This will identify neuronal groups that show altered excitability with activation or inhibition of astrocytes. In complementary studies of Aim 1b, we will express membrane-tethered versions of NKT in all known sleep-regulating neuronal groups to identify those subject to modulation by the glial protein. NKT is a secreted astrocyte protein, but this subaim will utilize neuronal expression of membrane-tethered NKTs to identify its cellular targets. Together, Aims 1a and 1b will identify sleep-regulating neurons that are subject to astrocyte modulation. Aim 2 will use Translating Ribosome Affinity Purification (TRAP) methods combined with genome-wide RNA-seq to test the hypothesis that the gene expression profile of sleep-regulating neurons is altered by astrocyte modulation. The studies of this aim will focus on identification of neuronal factors (receptors, intracellular signaling components) with vertebrate orthologs that may mediate the astrocyte modulation of sleep. Preliminary results documenting feasibility for the aims are included in the proposal. Together, the two aims will provide valuable information about neuronal targets of astrocyte modulation and identify neuronal molecular pathways that respond to astrocyte signals. Such pathways may be important for glia-to-neuron communication or more directly the regulation of sleep.