David J. Anderson - US grants

The long-range goal of this laboratory is to understand the cellular and molecular mechanisms that underlie the development of phenotypic diversity and plasticity in the mammalian peripheral nervous system. To simplify this complex problem we have focused on a particular sublineage of the neural crest, the sympathoadrenal lineage, which gives rise to adrenal medullary chromaffin cells and sympathetic neurons among other derivatives. Adrenal chromaffin cells are phenotypically plastic in that they can convert into sympathetic neurons in response to NGF. This plasticity is of potential medical relevance 1) because of its probable mechanistic relationship to at least some forms of nerve regeneration; and 2) because of the potential utility of using chromaffin cells or cells at earlier stages in the lineage in transplantation therapy for Parkinson's disease. The specific aims of this application are to I) Study the differentiation of committed embryonic sympathoadrenal progenitor cells in culture using defined populations isolated with specific monoclonal antibodies and fluorescence-activated cell-sorting. The responses of these progenitors to various growth factors and other signals will be analyzed. New progenitor cell lines we have produced may permit us to examine the molecular basis for some of these responses; II & III) Examine the molecular basis of chromaffin cell plasticity in terms of the expression of a neural-specific marker gene, SCG1O. We will explore the use of features of active or potentially-active SCG1O chromatin structure as a molecular marker of plasticity and developmental potential in this lineage. The regulatory elements that control expression of this gene durinq development, chromaffin cell plasticity and regeneration will be studied using transfected cell lines and transgenic mice; IV) Identify the immediate precursor to the committed sympathoadrenal progenitor. An in vitro system has been established in which this postulated "pre-progenitor" should commit to the sympathoadrenal lineage. we will find monoclonal antibodies that mark this preprogenitor, and make a cell line from this precursor as a first step towards identifyinq molecules involved in the commitment event. A novel "surface-tagging" approach will ba taken in an attempt to mark this cell, in which a promoter specific to this cell is use to drive the expression of a viral cell surface glycoprotein, in transgenic mice.

This proposal is for funds to purchase a new fluorescence- activated cell sorter for a multiuser facility in the Biology Division at Caltech. The sorter will be employed in a variety of projects, including sorting of immature cells for developmental lineage analysis, selection of mutant cells and transfectants, quantitation of cell-surface receptor expression in structure- function analysis, and multiparameter phenotypic analysis. Other applications include determination of cell cycle parameters in studies of cell cycle-linked gene expression. The facility currently operates a 1981 Ortho Sorter, which is overbooked, technically inadequate for many current applications, and no longer supported by the manufacturer's service representatives. The lack of a state-of-the-art sorter has become a sever bottleneck for many projects in the division.

Description: The goal of this project is to develop a new molecular method, termed recombinase-based lineage tracing" (RBLT), to provide a fate map of embryonic precursor cells that express the MASH-1 gene, a nuclear regulatory gene of the basic helix-loop-helix (HLH) family. Recombinase mediated lineage tracing provides a means to mark the mitotic progeny by DNA rearrangement driven by a site-specific recombinase (Cre). A primary advantage of this approach is the access to neural precursor cell populations that would likely be inaccessible to dye labeling or retroviral marking methods. Since the review of the original application, several significant advances have been made. First, the investigators demonstrated that Cre-mediated recombinations of a lacZ reporter gene are feasible for fate mapping experiments, shown by crossing activator mice expressing a CMV-Cre construct to transgenic animals harboring a reporter gene, chick actin XSTOPX lacZ. The offspring generated showed no developmental defects, thus offsetting a concern raised in the previous review. Second, the investigators will utilize the green fluorescent protein (GFP) as an alternative to alpha-galactosidase. Finally, a recent publication describing a null mutation in the MASH-1 gene demonstrated an essential role for MASH-1 in the development of several classes of neurons, including the sympathoadrenal lineage and olfactory neurons. The Cre-recombinase methodology proposed should facilitate studies on other neural lineages that express MASH-1, e.g., progenitor cells in the forebrain.

laboratory mouse

DESCRIPTION (Applicant's abstract): The identification of genes with brain subregion- and/or neuron subtype-restricted patterns of expression is a central goal of Molecular Neuroscience in the post-genome era, for several reasons. First, such information is critical in order to attach meaning to the quantitative data that will be obtained by massively parallel analysis of gene expression in the brain under various normal and pathological conditions. Second, it is important to identify genes, or sets of genes, that can be used to mark, functionally manipulate, and map the connections of, specific brain regions or neuron subtypes, in order to experimentally define the neuroanatomical substrates of behavior and brain disease. Third, the identification of such genes should yield not only valuable markers, but also subjects for functional genetic studies of brain and behavior. The goal of this project is to determine the feasibility of using microarrays to identify brain region- and/or neuron subtype-restricted patterns of gene expression in the murine central nervous system. Using Affymetrix chips and flexible content cDNA and 50-mer oligomer microarrays, we will compare patterns of gene expression across five structurally and functionally distinct adult brain regions (amygdala, hippocampus, olfactory bulb, cerebellum and peraqueductal gray). Genes exhibiting differential expression between these regions will be validated by in situ hybridization. The strategy is to carry out an initial analysis using the amygdala as the reference region against the other four test regions, validate selected clones by in situ hybridization, adjust the stringency of the search algorithms based on the results of these initial data and iterate the process until all of the candidates have been tested. Once the algorithms have been optimized, the procedure can be repeated for each of the other four brain regions. To facilitate this analysis, we will develop and apply a series of algorithms that take into account structural information (sequence motifs) in adjusting stringency criteria for analysis of microarray data. Finally, we will extend a prototypic object-oriented database to store, manage and access both quantitative and spatial information about gene expression patterns in the brain. At the end of this exercise, we should have accumulated both a comprehensive data set that will indicate which microarray technology and search algorithms are most effective for this type of endeavor, an initial collection of clones and expression patterns that should be of interest to the community, and a system for the management, storage and accessing of brain in situ hybridization data that may achieve widespread use.

The Gordon Conference on Neural Development has evolved into one of the key meetings in the field. The subject of developmental neurobiology is very complex, requiring integration of hypotheses and information at the molecular, cellular, and systems levels. The conference is intended to bring together such a mixture of research groups in a format highly conducive to both formal and informal exchange. The emphasis will be on the discussion of cutting-edge, unpublished research. In addition, the breadth of the meeting provides an excellent opportunity for those who are beginning their careers or moving into a new subject area. The meeting is small (approximately 125 participants); however, the Chair and Vice-Chair will strive to ensure that a diverse group of both junior and senior investigators attends. The financial support requested will also be used to increase the numbers of women and minorities participating in this meeting. The speakers we have chosen represent not only some of the most active groups, but also individuals with the capacity to generate useful discussion of their own and other topics. The concept of the meeting has been not to try to cover the entire field thinly, but to focus on areas of exceptional activity or promise. Featured in this year's program are the following topics: activity vs. genetic specification in the formation of neuronal connections, identity of stem cells in the adult brain, control of the neuron/glia fate choice, neuronal polarity, expression and functions of neuronal cadherins, axon guidance, transcriptional codes in neuronal identity, and development of sensory systems. Within these topics, we have included several speakers whose research has important clinical applications. The meeting is well-balanced, containing both promising young investigators as well as more senior leaders in the field. Forty-five to fifty minutes will be allowed for each speaker's topic, of which one- third will be devoted to discussion. The afternoons are open for informal interactions. Several poster sessions in which conferees can present their work will be scheduled; these have been extremely well attended at previous conferences. Most participants will be expected to present a poster, thus this meeting will serve both a scientific and training function.

This program will provide predoctoral training of students preparing for research careers in Molecular, Cellular, and Systems Neuroscience. It involves 26 faculty members from the Biology, Physics, Chemistry, and Engineering Divisions. It is a continuation of a program previously supported by NIH. Some research areas of special emphasis are: 1) neural development (control of cell fate, axon guidance and synaptogenesis in a variety of systems); 2) signal transduction mechanisms in neurons (sensory processing in the visual and olfactory systems of vertebrates and invertebrates, and synaptic transmission and plasticity in hippocampal neurons); 3) behavior (simple and complex behaviors in vertebrates, arthropods, and nematodes); 4) computational neuroscience (studies of single neurons, system of neurons, and whole organisms). The major components of our training activities are: 1) each student's individual research program under one or more faculty sponsors; 2) an organized curriculum of graduate courses; 3) preparation for qualifying examinations; 4) teaching activities; 5) an extensive and wide-ranging seminar program. Support is requested for 16 predoctoral trainees, who will be admitted to graduate study for a Ph.D. in Biology or in Computation and Neural Systems. Criteria for admission into the program include a strong motivation for a career in research and high quantitative ability. Our expectation that trainees will continue into productive research careers is supported by the records of previous trainees. Caltech has a strong commitment (at both the institutional and the Divisional levels) to increasing the representation of minorities in science. In the Biology program, we have made special efforts to attract exceptionally talented students from under-represented minority groups, and we have been quite successful in this effort in recent years. A number of these students are primarily interested in neuroscience research. The training faculty are located within several building clustered near each other on the Caltech campus. Multi-user facilities include DNA sequencing, oligonucleotide synthesis, peptide synthesis, protein expression and purification, monoclonal antibody production, a transgenic and 'knockout' mouse facility, and the Biological Imaging facility.

DESCRIPTION (Verbatim from the application): The transmembrane ligand ephrinB2 is specifically expressed by arterial but not venous endothelial cells in both embryos and adults, whereas its receptor EphB4 is conversely expressed by veins but not arteries. Both the ligand and receptor are essential for embryonic cardiovascular development. The objective of this proposal is to understand more precisely the function of ephrinB2-EphB4 signaling in angiogenesis. Using conditional loss-and gain-of-function manipulations in knockout and transgenic mice, we will test the hypothesis that bi-directional signaling between arteries and veins mediated by this ligand-receptor pair is essential for angiogenesis, and ask whether the cellular function of this signaling is primarily attractive or repulsive. In Specific Aim I, we will knock out ephrinB2 specifically within endothelial and endocardial cells, to determine whether its essential function is indeed exerted within the circulatory system. These studies will be complemented by experiments to selectively rescue the ephrinB2 knockout phenotype within the circulatory system. Temporally controlled, pan-endothelial knockout of ephrinB2 will also be performed, to determine whether the gene is also required at later stages of angiogenesis. In Specific Aim II, we will use both in vitro embryoid body assays of blood vessel formation and in vivo embryo chimeras in conjunction with heterozygous and homozygous ES cells mutant for ephrinB2 or EphB4 to distinguish whether the primary requirement for these genes in early cardiovascular development is in the heart, the peripheral vasculature, or both. In Specific Aim III we will perform constitutive and/or conditional pan-endothelial expression of ephrinB2 and EphB4 transgenes to determine whether the arterial- and venous-specific expression, respectively, of these genes is essential for their proper function in the circulatory system. In Specific Aim IV we will create lines of "dual-indicator" mice that can be used to simultaneously distinguish arteries and veins using genetically encoded photochemical markers. These mice will be used in both in vivo and in vitro experiments to further study the role of cell-cell interactions between arteries and veins and the role of ephrinB2-EphB4 signaling in mediating these interactions. Mechanistic studies of the role of ephrinB2-EphB4 signaling in development are highly like to inform our understanding of its function in adult angiogenesis, and may suggest new therapeutic strategies for the inhibition or promotion of angiogenesis in clinical settings such as cancer and heart disease, via pharmacological manipulation of these artery- and vein-specific signaling molecules.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the cellular and molecular mechanisms that control cell fate determination in the developing vertebrate nervous system, with a particular emphasis on the choice between neuronal and glial fates. Specifically, we propose to study the function and regulation of Olig1 and Olig2, two basic helix-loop-helix (bHLH) transcription factor genes we have recently isolated that are specifically expressed in the oligodendrocyte lineage (Zhou et al. (2000) Neuron 25:331). In Specific Aim I of this proposal, we will determine whether the Olig genes are essential determinants of the oligodendrocyte fate in vivo, by generating Olig1 and 2-deficient mice and analyzing the phenotypes of various allelic combinations of these mutations, using an extensive battery of molecular markers. In Specific Aim II, we will determine whether expression of Olig genes marks commitment to the oligodendrocyte lineage, by using vital markers incorporated into the gene targeting cassettes to isolate Olig1- and Olig2-expressing cells by flow cytometry. The developmental capacities of these isolated cell populations will be tested by in vivo transplantation and by in vitro cell culture. In the second part of this proposal we will investigate the regulation of Olig gene expression and its functional interactions with other determinants of neural cell fate in the spinal cord. The expression of Olig genes defines a restricted zone from which oligodendrocyte precursors will emerge several days later. In Specific Aim III, we will carefully map the relationship of the Olig gene expression domain to the domains of expression of other known regulators of spinal cord cell fate, using double- and triple-labeling with antibodies and/or cDNA probes and laser scanning confocal fluorescence microscopy. These latter regulators include components of a recently identified homeodomain code that specifies different progenitor domains (Briscoe et al. (2000) Cell 101:435); neurogenic bHLH factors and Notch ligands. In Aim IV, we will functionally test hypotheses for the regulation of Olig gene expression suggested by correlative data obtained in Aim III, using retrovirus- or electroporation-mediated gene transfer to mis-express candidate regulatory genes in the chick spinal cord and determine their effect on the domain of Olig gene expression, and vice-versa. An understanding of the cellular and molecular mechanisms that control oligodendrocyte lineage determination in neural progenitor cells is an essential prerequisite to the controlled manipulation of such cells for transplantation therapy of neurological diseases such as Multiple Sclerosis.

The survival of an organism depends on its ability to respond appropriately to environmental stimuli. In higher organisms, this response is mediated through the function of the nervous system. To maximize adaptability, the nervous system must not only distinguish between qualitatively different stimuli, but also their intensities. Two major properties allow the nervous system to achieve this goal. First, the nerve impulses produced by a given stimulus travel via a specific neuronal network. Second, the threshold and frequency of the nerve impulses generated by a given stimulus can be modified by external conditions. The information necessary for building both the neuronal network and its components for nervous impulse generation are encoded by the genome of the organism.
One of the major intellectual challenges of modern biology is to understand how this genomic information is translated to an appropriate axonal network, and how those networks function to produce appropriate behavior.
Genetic analysis of behavior is a powerful approach to these problems. It is typically carried out in three stages. First, a paradigm is designed in which the behavior of interest can be easily observed and scored in normal individuals. The second stage uses mutagenesis to identify mutant genes that show clear deviations in behavior. Third, modern genetic techniques make it possible to either shut down or activate specific regions of the nervous system, to identify the circuits within the neural network that produces the behavior.
The purpose of this project is a genetic analysis and silencing of specific neuronal circuits to examine two behavioral paradigms, the Drosophila models of pain and fear, that the investigator's group has developed. Both of these involve basic neuronal elements, including sensors that detect the stimulus and neuronal networks that execute the behavior when the sensor is activated.
Using Drosophila as a model organism, this research will increase the scientific understanding of the mechanisms that allow complex behaviors to be encoded by the genome,. The basic principles learned from these studies will provide a foundation for understanding the molecular and neural principles that underlie behavior in humans and other organisms. It is expected that students at a range of levels, from high school to graduate school, will participate in this research.

DESCRIPTION (provided by applicant): The objective of this proposal is to develop improved methods for inducible genetic marking, mapping and manipulation of specific brain regions or neuronal subpopulations in the mammalian brain. In Aim I, methods for isolating brain region-restricted and cell type-specific genes utilizing laser-capture microdissection together with DNA microarray analysis will be developed and optimized. These experiments will focus on identifying markers for limbic system structures, including the amygdala, BNST, hypothalamus and lateral septum. In Aim II, the utility and efficacy of different genetically encoded primary axonal markers will be compared, and genetically encoded, inducible anterograde and retrograde trans-neuronal tracers will be developed and tested in vivo. Proof-of-principle will first be established in the PNS using a well-defined model system, and then extended to the CNS. Aim III encompasses the development and in vivo testing of two alternative combinatorial, positive coincidence-detection systems for achieving region- or cell subtype-specific control of heterologous gene expression (e.g., neuronal tracers or silencers), without having to identify specific transcriptional enhancer elements for such regions or cells. One method is based on inducible site-specific DNA recombination. The other method is based on a "two-hybrid" system for inducible transcriptional activation. In both cases, expression of the reporter/tracer gene is induced only in cell populations in which the expression of two different "co-driver" genes overlaps. This "Venn diagram" approach allows different pairwise combinations of driver genes to be used to express reporter or tracer genes in only a restricted subset of the regions in which each individual co-driver gene is expressed. These methods will initially be developed, tested and optimized using highly specific genes with known overlapping patterns of expression in specific subsets of pain-sensing primary sensory neurons. In Aim IV, based on the outcome of Aim III, either the recombination-based or two-hybrid system will be selected for extension to the CNS, using an overlapping pair(s) of limbic system-specific genes identified in Aim I. In addition, the recombination-based system will be extended to permit activity-dependent trans-neuronal tracing of neurons expressing a specific marker gene. The combination of new limbic system-specific molecular markers and genetically encoded tract-tracing and neuronal ablation methods should improve our understanding of the functional neuroanatomy of emotion and affective disorders such as anxiety and depression.

[unreadable] DESCRIPTION (provided by applicant): This program will provide predoctoral training of students preparing for research careers in Molecular, Cellular, and Systems Neuroscience. It involves 25 faculty members from the Biology, Physics, Chemistry, and Engineering Divisions. It is a continuation of a program previously supported by NIH. [unreadable] [unreadable] Some research areas of special emphasis are: 1) neural development (control of cell fate, axon guidance, and synaptogenesis in a variety of systems); 2) signal transduction mechanisms in neurons (sensory processing in the visual, auditory, somatosensory and olfactory systems of vertebrates and invertebrates, and synaptic transmission and plasticity in hippocampal neurons); 3) behavior (simple and complex behaviors in vertebrates, arthropods, and nematodes, including behavioral genetics); 4) computational neuroscience (studies of single neurons, systems of neurons, and whole organisms). [unreadable] [unreadable] The major components of our training activities are: 1) each student's individual research program under one or more faculty sponsors; 2) a required course and an organized curriculum of elective graduate courses; 3) preparation for qualifying examinations; 4) teaching activities; 5) an extensive and wide-ranging seminar program; 6) regular presentations by students on their research progress; 7) a Neuroscience Retreat designed to foster intellectual crossfertilization among trainees. [unreadable] [unreadable] Support is requested for 16 predoctoral trainees, who will be admitted to graduate study for a Ph.D. in Biology or in Computation and Neural Systems. Criteria for admission into the program include a strong motivation for a career in research and high quantitative ability. Our expectation that trainees will continue into productive research careers is supported by the records of previous trainees. [unreadable] [unreadable] Caltech has a strong commitment (at the Training Program, Divisional and Institute levels) to increasing the representation of minorities in science. In the Biology program, we have made special efforts to attract exceptionally talented students from underrepresented minority groups, and have been quite successful in this effort in recent years. A number of these students are primarily interested in neuroscience research. [unreadable] [unreadable] The training faculty members are located within several buildings clustered near each other on the Caltech campus, including the newly-completed Broad Center for the Biological Sciences. Multi-user facilities include the Biological Imaging facility, a transgenic and 'knockout' mouse facility, a new fMRI facility located in the Broad Building, and facilities for DNA sequencing, peptide synthesis, protein expression and purification, monoclonal antibody production, electron microscopy and flow cytometry. [unreadable] [unreadable]

Chronic pain is a serious health problem that has remained largely refractory to therapeutic intervention. The development of new pain therapeutics would be aided by a better understanding of the molecular and cellular mechanisms mediating nociception. The Mas-related genes (Mrgs) are a recently discovered, large family of G-protein coupled neuropeptide receptors (GPCRs) that are expressed with exquisite specificity in highly restricted subsets of nociceptive sensory neurons. The goal of this Program Project gram is to mount a concerted, interdisciplinary effort to understand the molecular function of differem Mrgs, the function of the neurons that express them, and the nature of the circuits in which these neurons participate. The project integrates the efforts of three laboratories with complementary expertise. The laboratory of David Anderson, which discovered the Mrgs, will utilize state-of-the art methods of mouse molecular genetics to generate and analyze strains of mice in which different Mrg genes have been deleted, and in which Mrg-expressing neurons can be inducibly ablated or silenced, or their second- and higher-order projections traced. These mice can also be used to prospectively identify Mrg-expressing neurons for physiological and molecular genetic analyses. The laboratory of Allan Basbaum is experienced in the behavioral, neuroanatomical, physiological and pharmacological analysis of nociception, and will collaborate with Anderson's group to thoroughly characterize the phenotypes of mice lacking different Mrg genes, or Mrg-expressing neurons, as well as in the analysis of Mrg synaptic connectivity. Because all Mrg-expressing cells are contained within the IB4-positive subset of nociceptive neurons, this project dovetails with the Basbaum laboratory's ongoing interest in understanding the function of this subpopulation in pain. The laboratory of Melvin Simon has expertise in the molecular genetic analysis of signal transduction by GPCRs and G-proteins. They will apply this expertise to characterize the pharmacology and mechanism of action of Mrgs, as well as to identify both endogenous and surrogate ligands for these receptors. In vitro culture of Mrg-expressing neurons will be employed to analyze and mechanistically dissect the influence of different candidate Mrg ligands, and idemify components of the intracellular signaling circuit. These studies may eventually lead to novel Mrg-based therapeutics for the treatment of pain in humans.

The Mouse Core plays a central role in this proposal, providing for the breeding and management of the large colony of transgenic and knockout mice that will be generated for this project. Five different knockout strains have been or will be created for this project, and these will be each intercrossed with one another, as well as with 3-4 different strains of transgenic lines (tetO-responder lines), to generate multiple substrains. These animals are the source for all of the behavioral, physiological, pharmacological, anatomical and cell-based studies in the Program Project, and are a primary shared resource for all of the collaborative experiments among the three participating laboratories. The need for this core is justified by the large numbers of genetically modified mice that must be generated and maintained solely for the purposes of this project (see Budget Justification). The behavioral phenotyping experiments in particular require large numbers of animals of each strain and genotype, because the greater inherent variability of such experiments requires large numbers of animals to achieve statistical significance. The Mouse Core will leverage the infrastructure, personnel, equipment and other resources available in the Transgenic Animal Facility at Caltech (TAFCIT), while providing exclusive support for the generation, maintenance, breeding and shipping of transgenic and knockout mice strains associated with this Program Project.

In this proposal we will use molecular genetic methods to dissect the function of Mrgs, and the circuitry and function of the neurons that express these GPCRs. Using knockout mice expressing fEGFP from the MrgA1 and MrgD loci, we will ask: 1) what are the peripheral and central projection targets of MrgA1+ and MrgD+ neurons?; 2) Do MrgA and MrgD play a role in the peripheral or central targeting of sensory axons?; 3) Do MrgA and/or MrgD exhibit allelic exclusion? In collaboration with Allan Basbaum, we will ask: 4) How are MrgA1 and MrgD expression regulated in chronic inflammation and nerve injury?; 5) What are the behavioral consequences of loss of MrgA and MrgD function on nociception? In collaboration with Mel Simon, we will ask 6) How do ligands for MrgA affect the physiology of MrgA-expressing neurons in vitro, and are these effects dependent on MrgA function? In collaboration with Basbaum, we will ask whether deletion of the receptors affects the behavioral response to MrgA ligands in vivo. Using a tet-dependent transcriptional activator (rtTA) expressed from the MrgA and MrgD loci, and a series of tet-inducible responder mice, we will map the second-order neurons that receive afferent input from MrgA- and MrgD-expressing neurons, and third- or higher-order neurons to which they project. In collaboration with Basbaum, we will additionally investigate 2) the behavioral consequences of killing MrgA- and MrgD-expressing neurons at different stages of postnatal development, and 3) the behavioral consequences of reversibly silencing MrgA- and MrgD-expressing neurons. We will generate similar targeted mutations in MrgC11 and MrgB4/5, and ask an analagous series of questions about the function of these genes and of the neurons and circuits in which they are expressed. Using MrgC11 and MrgA knockout mice, we will delete the MrgAC gene complex and examine its functional consequences. Finally, we will exploit the specificity of MrgD expression to compare the gene expression profiles of isolated MrgD+, IB4+ and MrgD-, IB4+ nociceptive neurons. These studies may identify 1) additional genes involved in MrgD-related signaling; and/or 2) markers for other subpopulations of IB4+ neurons will different end-organ specificities. Together, these experiments should provide new insight into molecular and cellular mechanisms of nociception, and may ultimately lead to the development of novel pain therapeutics.

One of the great challenges facing modern biology is understanding how the brains of animals coordinate simple motor acts into complex behaviors. For example, the act of a fly leaping from a table and landing on the ceiling requires an intricate sequence of leg and wing motion rapidly modified by sensory feedback. How brains, even those as simple as a fly's, flawlessly execute such feats remains unknown. This project will combine advances in genetics and engineering to experimentally control the activity of individual neurons within the brains of animals that are behaving normally, thus allowing scientists to directly observe the behavioral consequences of specific brain circuits. This multidisciplinary research will involve three components. First, researchers will engineer ion channels within individual neurons that can be opened and closed with pulses of light, creating a switch that turns brain cells on and off. Second, they will genetically engineer animals with these controllable neurons. Finally, they will use sophisticated electronic devices, such as virtual reality flight simulators, that measure changes in behavior resulting from the experimental manipulation of specific brain circuits. This will allow the investigative team to map and decipher brain regions responsible for the control of various behaviors.

The research will be conducted on fruit flies, an important laboratory organism that is used in a wide range of genetic and medical research and is crucial to studying a wide variety of human diseases including alcoholism, senility and obesity. The work will also be incorporated into efforts to 'reverse engineer' flies and combine information about the brains, bodies and behavior of flies to create autonomous flying robots. An educational training and outreach program will foster the development of students and young scientists broadly skilled in biology, math and engineering and capable of approaching formerly intractable problems that require interdisciplinary approaches. This project is a collaboration led by California Institute of Technology (Michael Dickinson) and University of California-Berkeley (Isacoff).

DESCRIPTION (provided by applicant): Psychiatric disorders, such as PTSD, depression and generalized anxiety disorder, are increasingly being recognized as dysfunctions of specific brain circuits, rather than alterations in global "brain chemistry." In order to develop new therapeutic approaches based on an understanding of underlying disease mechanisms, it is necessary to understand the normal function of the affected circuits. In this application, we propose to apply new, genetically based, techniques for manipulating neuronal function and mapping neuronal connectivity, to dissect the microcircuitry that underlies conditioned fear and its extinction. Our focus is on understanding the function of subpopulations of interneurons located in the central nucleus of the amygdala (CeA), a brain region involved in emotion. One subset of these neurons is marked by expression of protein kinase C-4 (PKC-4). Our preliminary data indicate that genetically based inactivation of these neurons enhances conditioned freezing, suggesting that these neurons may normally act to gate output from CeA. Using a recently developed genetic system for neuronal silencing, based on an ivermectin (IVM)-gated chloride channel, and an "intersectional" strategy to target expression of this heteromeric channel exclusively to PKC-4 cells in CeA, we will test this hypothesis and investigate the functional role of these neurons in fear learning and fear extinction, as well as in unconditional fear and anxiety (Specific Aim I). In Specific Aim II, we will further investigate the role of these neurons using neuronal activation strategies based on light (channelrhodopsin-2) or chemical activation. These experiments will test the necessity and sufficiency, respectively, of PKC-4 neurons in emotional behaviors mediated by the amygdala. In Specific Aim III, we will map the inputs and outputs to and from these neurons, using genetically based neuronal tracing and electrophysiological techniques. Finally, in Specific Aim IV we will test the hypothesis that activation of PKC-4 neurons is required for the behavioral effects of anxiolytic drugs, such as benzodiazepines. These studies should begin to provide a functional dissection of the amygdala at the level of granularity of specific neuronal subtypes, and may identify new cellular targets for therapeutic intervention in psychiatric disorders. PUBLIC HEALTH RELEVANCE: Psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), exact a significant toll on public health, yet current methods to diagnose and treat them are inadequate. In order to develop a new generation of more effective treatments for these illnesses, with fewer side-effects, it is necessary to identify the underlying brain circuits that are impaired, understand the normal function of these circuits in emotional behavior, and describe how this function is altered in a given disorder. The present proposal applies an arsenal of new, genetically based, tools for dissecting neural circuit function at a level of specificity that has not previously been achieved, to understand the 'gating'mechanisms that control the flow of information through the amygdala, a brain structure important in learning (and "unlearning") fear.

DESCRIPTION (provided by applicant): Affective disorders, such as bipolar disorder, schizophrenia and PTSD, as well as drug and alcohol addiction, are increasingly being understood as dysfunctions of specific brain circuits. In order to develop new, rational therapeutic approaches to these illnesses, it is necessary to understand the normal function of the disrupted circuits, and how they are altered in a given disorder. Central to this objective is the use of molecular genetic-based tools, in the mouse, to mark, map and manipulate neural circuitry. In this application, we propose to develop new, genetically based, techniques for mapping the connectivity of neurons that are activated by a specific stimulus or during a specific behavior. Specifically, our goal is to fill two lacunae in the current "toolkit" for genetic mapping of neuronal circuits in mice: a conditional, viral-based method for anterograde trans-neuronal tracing;and a method to stably and efficiently mark neurons that have been transiently activated. In Specific Aim I, we will develop recombinant variants of the Herpesvirus strain H129, which is transported trans-neuronally in the anterograde direction, using a homologous recombination-based method we have developed. These variants will be dependent on Cre recombinase for expression of a marker and/or replication. We will test these recombinant strains in several lines of Cre-expressing mice, in both the peripheral and central nervous system. In Specific Aim II, we will develop and test two methods for stable, activity-dependent marking (SADM) of neurons, both of which are based on the expression of Cre recombinase. These methods are based on the combined use of transgenic mice and stereotaxically injected, Cre-dependent recombinant viruses. They allow, in principle, the expression of any marker or effector gene in transiently activated neurons. In Specific Aim III, we will combine the technologies of Aims I and II, to achieve activity-dependent retrograde and anterograde trans-neuronal tracing of neural circuits. Finally, in Specific Aim IV, we will modify the methods of Aim II, to permit selective stable, activity-dependent marking of GABAergic neurons (SADM-GAD). This will permit the visualization of active inhibitory networks in the brain, and will open the way to marking and manipulating them in an activity-dependent manner. The methods described in this application will make available to the community a new set of tools that should have wide applications in neuronal circuit tracing, in both normal mice and disease models. PUBLIC HEALTH RELEVANCE: The development of new treatments for psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), as well as for drug and alcohol addiction, will require a better understanding of the brain circuits whose functions are disrupted in these disorders. The present application is aimed at developing new, molecular genetically based, methods for marking, mapping and manipulating neural circuits in the mouse, the best genetically accessible system for modeling such disorders. These methods should have wide applicability in furthering our understanding of the neural circuit basis of affective disorders and addiction in this important model system, and may identify new cellular targets for the development of novel therapeutics.

DESCRIPTION (provided by applicant): Arousal involves a state of heightened neural activity and lower threshold sensitivity to environmental stimuli. Substance abuse (SA) often involves the uncontrollable self- administration of drugs that alter levels of arousal. Epidemiological and genetic data suggest a linkage between SA, sleep disturbances and arousal/attentional disorders such as ADHD. Thus an understanding of the genetic and neural circuit-level mechanisms that control arousal may lend insight into the pathophysiology and genetics of SA. A fundamental question is whether arousal is a unitary, generalized state, or whether there are different forms of arousal, controlling different behaviors. Consistent with the latter view, multiple neuromodulatory systems have been implicated in arousal, including biogenic amines (BAs), acetylcholine (ACh), hormones and neuropeptides such as orexin. Furthermore, recent data from my laboratory have shown that in Drosophila, even a single neuromodulator (dopamine;DA) can influence different forms of arousal (sleep-wake vs. stress-induced) in opposite directions, by acting through different neural circuits. These data suggest that a knowledge of the specific neural substrates on which different neuromodulators act to influence arousal, in different behavioral settings, will be essential for understanding the circuitry of arousal-related disorders, including addiction. In this proposal, we will address the following questions, using Drosophila as a model system: 1) Does DA influence other forms of arousal, and if so through what circuits? 2) Which other neuromodulators influence arousal, what form(s) of arousal do they control and through which circuits do they act? To address these questions, we will develop a general method to visualize the activation of neuromodulator receptors on specific neural circuits by their endogenous ligands in vivo. We will use this method to visualize the neural circuits that are regulated by different neuromodulators in different behavioral paradigms that are influenced by arousal. If successful, this method should be broadly applicable to a variety of genetically accessible model organisms and could transform studies of neuromodulation and its role in SA, addiction, attentional and hyperactivity disorders, and sleep disturbances. PUBLIC HEALTH RELEVANCE: Neuromodulators such as dopamine play a key role in substance abuse, ADHD and sleep disorders. All of these disorders are linked to disturbances in arousal. Whether there are different forms of arousal, each linked to different behaviors and behavioral disorders, and the role of different neuromodulators in controlling various forms of arousal, is poorly understood. This proposal aims to develop new methodology to systematically map the roles of different neuromodulators in different settings of arousal.

Fear is a powerful emotion and under normal conditions this works to the advantage of every species on this planet. One of the most poignant examples of this is post-traumatic stress disorder (PTSD). This project maps the neural circuit underlying the extinction of PTSD by combining molecular genetic tools with in depth behavioral analyses. Using a rodent model of PTSD adapted for use in the mouse, it extinguishes specific symptoms of PTSD such as enhanced fear, increased aggression and disruptions in sexual behavior, by genetically identifying and optogenetically targeting specific subsets of neurons known to be involved in fear inhibition. This project is comprised of three aims. Aim 1 determines whether extinction under the optogenetic activation of a population of cells in the lateral division of the central nucleus of the amygdala known to be involved in fear inhibition (PKC-d+ neurons) can subsequently reduce PTSD-induced enhancements in fear, increased aggression, and alterations in sexual behavior. Aim 2 determines whether optogenetic silencing of a distinct but intermingled population of neurons (CRH+ neurons) in the same brain region can produce the same reduction in PTSD symptomology. Aim 3 examines whether similar effects can be obtained using selective silencing or activation of prefrontal cortex projections to the amygdala.

This project has significant intellectual merit in its cross-disciplinary approach. Namely, it combines cutting-edgemolecular biology and genetic tools with well-designed behavioral paradigms. The application of such molecular techniques with solid
behavioral design and analyses are intellectually important in terms of the extent to which these tools can be applied to behavioral neuroscience, taught, and shared with the public.

Broader Impacts: The dissection of PTSD extinction circuitry has the powerful potential to provide a highly targeted, translational approach for the treatment of anxiety disorders and phobias. In addition, this project encourages the teaching, learning and training of optogenetics and applications of molecular biology to questions within psychological science from within the publically accessible and motivated framework of PTSD treatment.

? DESCRIPTION (provided by applicant): The brain circuit is an intricately interconnected network of a vast number of neurons with diverse molecular, anatomical and physiological properties. Neuronal cell types are fundamental building blocks of neural circuits. To understand the principles of information processing in the brain circuit, it is essential to have a systematic understanding of the common and unique properties for each of its components - the cell types, how they are connected to each other, and what are their functions in the circuit. From the study of numerous circuits, many types of mechanisms have been proposed regarding the roles of different cell types in signal processing. However, despite of the importance, we are far from a comprehensive understanding of the number and kinds of cell types in the brain or a given circuit. We do have a wealth of knowledge on the major cell types in each region, and many examples of specific types. But for the most part, due to the lack of systematic efforts, we don't know the complete cell type composition of most circuits, and we have very little idea about the degree of variation and heterogeneity among single cells, both within a given type and between different types. To address this issue, we propose to establish a comprehensive and standardized cell type characterization platform that can be scaled up to systematically examine the properties and function of cell type components in any neural circuits throughout the brain. To implement this, we propose a model for collaboration between academic labs/centers and Allen Institute for characterizing cell types in specific brain circuits, with all the QC-passed daa going into the Allen Institute Cell Types Database and becoming publicly available. We will test a range of experimental approaches, encompassing molecular, anatomical and physiological measurements and their integration at the single cell level. Our proof of principle studies are based on comparison of three major brain neural circuits in the mouse brain: two closely related cortical circuits - primary visual cortex (V1) and primary somatosensory cortex (S1), and a more distinct circuit - the hypothalamus/amygdala emotional pathway. These two axes of comparison should be very informative in assessing the reliability and generality of the cell type characterization approaches we will be testing. We thereby hope to determine the critical parameters and metrics necessary to classify neurons into discrete cell types, guided by their functions. Thus, we anticipate that this project and the resources it produces will have a broad impact and catalytic effect on the scientific community studying brain circuitry function and dysfunction.

? DESCRIPTION (provided by applicant): A common metaphor to describe the brain is that it is like a supercomputer. Consequently, current efforts at improving technologies for large-scale recording of brain function are primarily focused on measuring its electrical activity. However, unlike a supercomputer, the brain is an electrochemical machine. Superimposed upon its network of synaptic connections is a chemical connectome, a largely invisible network of neuromodulators, such as serotonin and neuropeptides (NPs), which exert a profound influence on brain function. Neuromodulators influence brain states that alter the computations performed by neural circuits, and are central to emotion, mood and affect. An understanding of neuromodulatory influences is therefore relevant to psychiatric disorders in humans. Without the ability to measure and manipulate the release of specific neuromodulators, our understanding of neuronal circuit function will be fundamentally incomplete. Nevertheless, a gap remains between our ability to monitor brain electrical activity, and our ability to monitor chemical activty with commensurate spatio-temporal resolution. Specifically, there is no method to visualize the release of specific NPs in the brain, at individual synapses. To fill this gap, we propose to develop a method for imaging the release of specific NPs at the level of nerve terminals, in vivo. The long-term goal is to develop new methods for visualizing, detecting and inhibiting NP release in vivo, and to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits. The overall goal of this proposal is to developa novel approach for time-resolved imaging of NP release from nerve terminals. The central objective of this proposal is to tag components of large dense core vesicles (LDCVs), and specific NPs, with pH-sensitive fluorescent proteins and to determine whether these reporters can be used to image neurosecretory granule release. Drosophila melanogaster provides a useful test-bed for this technology because of its genetic manipulability and sophisticated imaging methodologies. To achieve our objective, in Aim 1 we will fuse different pH-sensitive fluorescent reporters to the protein coding sequences of several LDCV-specific proteins and NPs, and generate transgenic flies. In Aim 2, we will use optogenetic activation of specific neuropeptidergic neurons containing these reporters in vivo, to determine whether activation-dependent increases in fluorescence can be detected, and distinguished from synaptic vesicle (SV) release. The contribution will be to determine the feasibility of the proposed approach, and to achieve a proof- of-principle application of the method. This contribution is significant, because it has the potential to create a transformative new technology with broad general applications. The contribution is innovative, because it combines expertise from cell biology, molecular genetics and neural circuit analysis to develop a novel methodology. The work proposed in this application will therefore benefit the field of neuroscience as a whole, and also enable studies of neural chemistry and circuitry whose dysfunction may underlie psychiatric disorders.

? DESCRIPTION (provided by applicant): Defining brain circuits that control decisions, such as those between social interactions and asocial behaviors is an important problem in neuroscience with high relevance to human health. These neural circuits are located in evolutionarily ancient brain regions, such as the amygdala. In humans, decreased social interactions are a key symptom domain in psychiatric disorders such as autism, and are thought to involve the amygdala. The amygdala is a complex structure consisting of at least 12 distinct subregions. The amygdala circuits that control conditioned fear in the basolateral and central amygdala have been intensively studied. However a gap remains between our understanding of these circuits, and those in different amygdala subnuclei that control social interactions. The latter are thought to be located in the medial subdivision of the amygdala (MeA). To fill this gap, we will begin to dissect the function of MeA circuits that control the balance between social interactions and repetitive self-grooming, an asocial behavior. This balance is important because disorders such as autism often feature increased repetitive asocial behaviors, as well as decreased social interactions. The long-term goal is to understand the circuit-level control of this balance at a brain-wide level. The overall objective of this application is to define the roleof different MeA neuronal subpopulations that antagonistically control social interactions vs. repetitive self-grooming, and to understand the circuitry through which this antagonism is exerted. The central objective of this proposal is to study how GABAergic and glutamatergic neuronal subpopulations in the medial amygdala reciprocally regulate these opponent activities. The rationale for this research is that it will reveal fundamental mechanisms of neural circuit function in a brain region that is relevant to human health. To achieve our objective, we will map the functional projections of vGAT+ and VGLUT2+ MeApd neurons that control social interactions vs. repetitive self-grooming, respectively (Aim 1); test the hypothesis that social interactions are controlled by a dis-inhibition circuit and map that circuit (Aim 2); determine the mechanism by which vGAT+ and vGLUT2+ subpopulations exert antagonistic control of social vs. self-grooming behaviors (Aim 3); determine whether the dual functions performed by each of these subpopulations derive from common or distinct cell types (Aim 4). The contribution will be to apply state-of-the- art genetically based tools to dissect circuit-level mechanisms in MeA that control social vs. repetitive asocial behaviors. This contribution is significant because it will oen up the study of amygdala circuitry controlling social interactions, at a level of cellular specificty that has not yet been achieved. The contribution is innovative, because it investigates novel features of amygdala circuitry that we have recently uncovered involving the excitation:inhibition balance. The work proposed in this application will therefore both advance our basic understanding of neural circuit functional organization, and shed light on the particulars of amygdala neuronal subpopulations and circuitry that may relevant to human disorders affecting social interactions.

Project Summary/Abstract Defining the brain mechanisms that mediate multidimensional representation of emotion states, such as fear, is an important problem in neuroscience with high relevance to human health, including psychiatric disorders such as anxiety and depression. The study of fear in animal models has been dominated by the Pavlovian fear conditioning paradigm, and a focus on the amygdala. However there is a need to extend the study of fear circuitry to extra-amygdala systems, as well as to paradigms for innate fear where emotion states can be studied without the additional complexities introduced by learning. There is also a need to expand the study of such circuits from a focus on single nuclei to meso-scale connectivity and function. The medial hypothalamic defensive circuit mediates innate defensive responses to predators. Recent data have identified neurons in the ventromedial hypothalamic nucleus (VMH) expressing the transcription factor SF1 as necessary and sufficient for defensive behavioral and autonomic responses to a predator. However little is known about the precise role of these neurons, and their targets, in representing threatening stimuli, and transforming this representation into emotion states and defensive responses. To fill this gap, we are using state-of-the-art tools for recording, imaging and perturbing neural activity in this system, using SF1+ neurons as a point-of-entry. Our broad, long-term objective is to understand how emotional stimuli are represented and transformed into internal states and behavioral responses. The central objective of this proposal is to determine how VMHdm/c SF1+ neurons, and associated circuitry, represent multi-modal threatening stimuli, and generate defensive responses. The rationale for this research is that the study of evolutionarily ancient brain circuits that control conserved emotion states such as fear is likely to yield general principles of multidimensional emotional representation. To achieve our objective, we will characterize how SF1+ neurons represent multi-modal threatening sensory cues (Aim 1); determine the relationship of neuronal activity in VMHdm/c SF1+ neurons to observable responses to threatening stimuli (Aim 2); investigate meso-scale circuit interactions controlling defensive responses by recording simultaneously from multiple regions during exposure to threatening stimuli (Aim 3); and investigate the circuit-level mechanisms underlying experience-dependent influences on acute responses to threatening stimuli (Aim 4). The contribution will be to apply state-of-the-art genetically based tools to study the representation of multimodal threatening stimuli and their causal functions. This contribution is significant because it will advance our understanding of the micro- and meso-scale circuit dynamics underlying emotional representations and responses. The contribution is innovative, because it represents the first time that this circuitry has been studied using such multidimensional systems-level approaches. The work proposed in this application will therefore increase our understanding of fundamental brain mechanisms of emotion representation, with potential relevance to understanding and treating human psychiatric disorders.

Project Summary/Abstract This proposal responds to an NIMH notice NOT-MH-18-036 aimed at the development and study of novel, computationally defined behavioral assays, and at applying theory and mathematical modeling to better capture the richness of complex, naturalistic behaviors. Specifically, we aim to develop novel computational tools for analyzing social behaviors in freely moving mice, and relating those identified behaviors to neural circuit activity in brain regions that govern the expression of those behaviors. Social behavior is affected in many human psychiatric disorders, such as autism, schizophrenia, and depression. We propose an interdisciplinary, collaborative approach to fill two major gaps that present a barrier to studies of social behavior: 1) the lack of quantitative and high-resolution descriptions of naturalistic social behaviors in freely moving animals, and 2) the difficulty of relating neural activity recorded in deep subcortical regions that govern such behaviors, such as the hypothalamus and extended amygdala, to animals' actions or to models of behavioral control. Our objective is to create a computational behavior analysis platform that integrates automated measurement of naturalistic social behavior, synchronous large-scale recording or imaging of neural activity, and apply these to a novel assay to investigate social behavioral decision-making. The central objective of this proposal is to extend our Mouse Action Recognition System (MARS) to create a platform that allows facile training of supervised and unsupervised behavior classifiers, quantitative correlation with simultaneously acquired neural recording or imaging data, and which can be flexibly adapted to additional behavior assays. The rationale for this approach is that fine-grained quantification of social behavior, and its correlation with neural recordings, is necessary to form and test theories of behavioral control by subcortical brain regions. While automated tracking and ?pose? estimation software such as DeepLabCut have made tracking of animals' body positions more feasible, the identification of social behaviors from pose data is a non-trivial problem, requiring a separate computational approach that takes into account the relative movements of multiple animals over time. To achieve our objective, we will broaden the palette of social behaviors MARS can detect using machine learning and generative models (Aim 1), develop methods to relate those behaviors to neural activity (Aim 2), and extend MARS to additional assays to study neural correlates of social decision-making. This contribution is significant because it will create a resource that will transform our ability to study micro- and meso-scale subcortical circuits controlling social behavior. The contribution is innovative because it combines expertise from circuit neuroscience and computer vision/machine learning to create new tools for understanding the link between neural activity and behavior, in a context that is relevant to understanding dysfunctions of neural circuits that underlie human psychiatric disorders.

Project Summary/Abstract This proposal responds to an FOA (RFA-NS-18-030) calling for 1) ?novel approaches to understand neural circuitry associated with well-defined social behaviors;? 2) Distributed circuits that contribute to the coordination of motivational states and reward behavior;? 3) ?Empirical and analytical approaches to understand how behavioral states are emergent properties of the interaction of neurons, circuits and networks.? The study of subcortical circuits that control conserved, naturalistic behaviors is crucial to understanding brain function. We aim to understand how dynamic interactions between different circuit nodes in the Hypothalamic-Extended Amygdala Decision (?HEAD?) network control innate social behavior decisions, e.g., between aggressive and reproductive behaviors. We propose an integrated approach to this problem that combines microendoscopic imaging (MEI) of genetically identified neuronal subpopulations with automated, machine learning-based classification of social behavior in freely moving mice, together with functional perturbations of neuronal activity in vivo. Our broad, long-term objective is to understand how distributed activity among interconnected structures in the HEAD network controls moment-to-moment decisions between competing goal-directed behaviors that are crucial for the survival of animals and humans. The central objective of this proposal is to understand how information flows through this network during social interactions, and is decoded to control the decision to engage in reproductive vs. aggressive social behaviors. To understand how activity in ?upstream? nodes controls neural representations in ?downstream? nodes, we will implement a novel approach combining reversible chemogenetic inhibition of the former with concurrent imaging of neuronal population activity in the latter. The rationale for this approach is that an understanding of the system requires characterizing the effects of functional manipulations on both behavioral and circuit-level phenotypes. To achieve our objective, we will first characterize the neural coding of behavior and conspecific sex identity in multiple nodes of the extended amygdala, using single-site microendoscopic imaging and computational analytic approaches (Aim 1); determine how perturbations in the activity of such nodes influence representations in hypothalamic nodes (Aim 2); investigate the roles of intra- and inter-nuclear interactions in determining the balance of activity between aggression and reproduction-promoting hypothalamic nodes (Aim 3); determine how this balance is decoded by downstream mid-brain structures to determine the type of social behavior to express (Aim 4). This contribution is significant because it represents a systems-level approach to understanding how a subcortical network controls behavioral decision-making. The contribution is innovative because it integrates analysis of neuronal population activity, quantitative measurement of naturalistic social behavior and functional perturbations of activity in specific neuronal subpopulations to gain insight into how distributed neural circuits control survival behaviors, in a context that is relevant to maladaptations causing human psychiatric disorders.