Susan M. Dymecki - US grants

The overall objectives of this project are to develop a binary recombination-based system to fate map and manipulate somatic lineages in the mouse embryo, and to utilize this new tool to reveal mechanisms underlying establishment of neural crest cell lineages. A central issue in mammalian development -understanding how lineage and environment interact to determine phenotype - has been limited by the inability to follow the fate of specific cells in situ and to observe the distribution of their progeny in the embryo throughout gestation. Such precise lineage mapping is prerequisite to understanding developmental determinants of cell proliferation, differentiation, and migration. Knowledge of these mechanisms is fundamental to understanding the origins of congenital malformations. We have engineered an in situ lineage marking system in transgenic mice that should mark specific populations of cells in a heritable, cell autonomous, non-diluting fashion during embryonic development. Additionally, this system can be used in conjunction with homologous recombination to perform lineage/tissue-specific in vivo mutagenesis. We have exploited an excisional recombination system found in yeast for these purposes: the recombinase FLP catalyzes recombination between direct repeats of FLP recombination targets (FRTs) excising the intervening DNA. Since initiating this project in 1993 we have: (1) constructed a modular set of FLP and universal target (FRT-disrupted lacZ) vectors; (2) demonstrated efficient FLP-activation of the target transgene in embryonic stem (ES) cells; (3) generated separate FLP and target (FRT-disrupted lacZ) transgenic mouse lines; and (4) demonstrated FLP-mediated recombination in embryos from a FLPxtarget cross. We are now in the unique position to fully characterize this system in vivo as a tool for marking cell lineages and for directed modifications of the mouse genome. Because neural crest cells migrate extensively and differentiate to form a variety of cell types they are optimal to study how lineage and environment interact to determine cell fate. Derangements of neural crest cell development are implicated in numerous congenital malformations including neural tube, limb, cranial, enteric ganglion and cardiac defects, deafness, and thymic agenesis. A major unanswered question is when and how does this population of cells generate such phenotypic diversity. Toward the proposed aims, we have generated transgenic mice expressing FLP in the dorsal CNS as a means to activate lacZ in neural crest progenitors. We will: (1) utilize this marking system to map the murine neural crest; (2) identify similarities and differences between the murine map and that of the chick, the latter providing much of our current knowledge; (3) move from descriptions of cell fate to analysis of underlying mechanisms through lineage studies in mutant embryos. Mechanisms of pathogenesis associated with Splotch (Pax-3), lethal spotting (Is), and W (c-kit) phenotypes will give insight into the pathogenesis of parallel human syndromes (e.g. Waardenburg Syndrome and Hirschsprung's disease).

Description: (Taken directly from the application) This project aims to delineate genetic mechanisms which specify pattern and shape of the endochondral skeleton by focusing on a newly identified skeletogenic locus called bdytg. Mice homozygous for the bdytg mutation show a reduction in size and number of appendicular long bones, and hence the name brachydactyly (bdy, or short digit). This mutation, generated by transgene insertion, presents a ready means to identify a new skeletogenic factor and provides a unique opportunity to study its interaction with known bone regulators. Because developmental processes involved in skeletal formation are reiterated later during adult bone remodeling and repair, we hypothesize that this new factor may help elucidate the pathogenesis of various human skeletal syndromes. Moreover, it should provide insight into the design of new therapeutics to influence adult responses to bone injury or disease. Skeletal pattern is established and integrated at many levels during embryonic development beginning with the condensation of chondrogenic cells, progressing to segmentation of these condensations to form new cartilage elements, culminating in their ossification to generate an articulating skeleton. Despite the importance of segmentation in determining both bone shape and number, relatively little is known about the genes controlling it. This application sets out to fill this gap by cloning a new segmentation gene (bdytg) and placing it within the context of factors known to control skeletogenesis (e.g., BMPs, growth differentiation factors (GDFs), Hedgehogs, PTHrP, and cognate receptors). Specific Aim 1 will identify the bdy gene and determine its function in chondrogenic growth and segmentation. Towards this aim we will: (1) identify the murine bdy gene (a candidate cDNA is already in hand); (2) determine the precise molecular nature of the bdytg mutation; (3) profile bdy expression during limb development; and (4) employ both gain-of-function and loss-of-function strategies to establish bdy function within the context of known skeletogenic signals. Specific Aim 2 examines chondrogenic segmentation in wild-type and bdy mutant limbs, using both embryologic and histologic assays, along with current molecular genetic methods. These studies will order the bdy gene relative to key skeletogenic pathways (e.g., BMPs, GDFs, Ihh, PTHrP) and will address the mode of action of the bdytg allele, determining whether it functions in a cell autonomous or non-autonomous fashion.

DESCRIPTION (provided by applicant): The broad objectives of this R21 RFA-DA-0 1-011 application are to extend the capabilities of the FLP-ERT and Cre-loxP site specific recombinase systems in mice by developing tools that will advance the following: 1) the ability to misexpress genes with greater temporal, spatial, and molecular precision, for example, in specific populations within populations of cells; 2) the ability to screen transgenics easily for recombinase activity, including FLP and Cre strains resulting from insertional mutagenesis; 3) the capacity to image aspects of cell morphology, while concomitantly relating this morphology to combinatorial patterns of gene expression; 4) the ability to determine cellular function (versus gene function) of specific cell populations. These capabilities are not presently available and would represent significant advancements. The studies described in this proposal concentrate on generating six widely useful recombinase target mouse strains that will extend the functionality of the many cell-specific FLP and Cre mice presently available. First, we will address the need for a high resolution dual responsive indicator mouse strain. While indicator strains exist that mark cells following either FLP- or Cre-mediated excision of disrupting DNA, no strains are presently available that achieve the higher resolution to specifically mark cells at the intersection of FLP and Cre expression domains (e.g. reflecting the overlap of two developmentally critical genes). Importantly, we will construct this dual responsive indicator strain using reporters designed to highlight cell morphology. From this dual responsive strain, we will go on to derive two broadly useful single, FLP or Cre, responsive indicator strains harboring properties not yet available. Second, a dual responsive ablation strain will be generated that allows for precise elimination of cell subpopulations in the embryonic and adult mouse. Development of a widely useful ablation strain will fill a significant technical gap by providing a powerful means to study the requirement for different cells in the development, structure, function, and aging of an organ system. From this strain we will go on to derive two broadly useful single, FLP or Cre, responsive ablation strains. Third, we will demonstrate the potential of the proposed six mouse strains by using them to extend our studies on the development and function of a brainstem system critical for movement control and sensorimotor transformations - the precerebellar afferent system. Because these novel mouse strains are designed to function in nearly all cell types, their impact on the generation of mouse models for human development and disease should be far-reaching.

DESCRIPTION (provided by applicant): The objective of this 2-year R21 application is to extend the capabilities of the Flp-FRTsite specific recombinase system (pioneered in mice by my laboratory) by generating inducible tools that will permit the generation of temporally controlled genetic modifications in embryonic and adult mice; notably, tools that can be used in parallel with either constitutive or inducible Cre-loxP-based reagents. This resulting dual recombinase capability is not presently available and would represent an important advance, permitting the engineering of either successive gene modifications as a means to model multistep developmental or adult disease processes or simultaneous gene modifications as a means to model multigene disorders. This advanced capability to temporally regulate recombination will be especially important for study of processes occurring at late embryonic and adult stages, those processes most limited by standard (non-inducible) conditional approaches. The proposed experiments focus on generating two types of inducible Flp tools, one broadly active and the other tissue-specific. These experiments follow from our previous results showing that the Flp variant, Flpe, is as effective in mice as Cre, and from the work of others showing that steroid-receptor fusions with Cre enable temporal regulation of recombination. In aim 1, we will introduce into mice widely inducible Flpe transgenes capable of catalyzing genetic changes in cells of diverse tissues at chosen times in embryonic and adult life. In aim 2, we will introduce into mice a tissue-specific inducible Flpe transgene capable of mediating genetic modifications in both a spatially and temporally regulated fashion in the neural tube. These mice will permit the genetic modification of specific subsets of cell lineages, distinguished by their temporal emergence from the dorsal neural tube. Moreover, they will serve as a prototype for other tissue-specific inducible Flpe transgenics. While the proposed tools will provide new capabilities for delineating processes critical to the development and health of many organ systems, our particular interest centers on brainstem lineages, in particular, those lineages that emerge successively from the rhombic lip region of the dorsal hindbrain. We plan to apply the proposed tools in conjunction with Cre-loxP-containing reagents to manipulate "late-born" rhombic lip lineages critical to the formation of both the precerebellar afferent system, a set of diverse nuclei essential for motor control, and the medullary raphe system, a center for homeostatic control in which developmental abnormalities have been implicated in sudden infant death syndrome.

DESCRIPTION (provided by applicant): Elucidating the pattern of connections between neurons is key to understanding nervous system function and dysfunction. Yet, conventional methods to delineate brain circuitry have been limited by their invasiveness, lack of reproducibility, and lack of cell specificity. Similarly limited are methods for modulating neuronal activity, specifically those methods essential for delineating relationships between particular circuits and perception or behavior. Our goal is to address these technical gaps by harnessing the power afforded by genetics -- we propose engineering transgenic mice bearing conditional alleles encoding transneuronal tracers, optical reporters, and/or neuromodulators that, in conjunction with the hundreds of region/cell-specific Cre and Flpe recombinase mice presently (and soon to be) available, will establish a set of enabling reagents for constructing functional connectivity maps of unprecedented resolution. The proposed genetic tools incorporate a high resolution dual recombinase (Cre and FIp)-mediated transgene activation paradigm pioneered recently by my lab in which selectivity can be improved by orders of magnitude relative to that achieved using conventional transgene (or even single recombinase) expression systems, while also providing great versatility. Using this system, tracer or modulator expression can be conditionally activated in as selective a group of neurons as possible so that circuits may be studied in virtual isolation; moreover, the tools are sufficiently versatile to permit any selected circuit to be studied at any stage. Using our strategy, in Aim 1 we generate three sets of transgenics for transneuronal tracing: one set for conditionally visualizing virtually any chain of neural connections in the direction of circuit flow (anterograde); a second set for delineating connections opposite to circuit flow (retrograde); and a third set (informed by the first two) for visualizing simultaneously both anterograde and retrograde connections. In Aim 2, we generate a similar set of selective, versatile, and conditional transgenics, but rather than having the capacity to trace connections, these tools permit neuronal silencing by suppressing either synaptic transmission or electrical conductance. In Aim 3, we validate the efficacy of these tools by applying them along with specific Cre- or Flpe-expressing mice to trace and modulate the well-defined neural pathways of the visual system. Additionally, all of these strains permit the simultaneous visualization of plastic changes in neuron morphology and facilitate integration of neurophysiological studies with the generated maps. Completion of these aims will provide tools that can be used by numerous investigators with different sets of recombinase-expressing mice thereby leveraging the work from this grant into a collective and comprehensive circuitry mapping effort.

DESCRIPTION (provided by applicant): Recovery Act Funds through the NIH Basic Behavioral and Social Science Opportunity Network (NOT- OD-10-O32) would enable us to incorporate, for the first time, fundamental behavioral research into an existing program (parent R21 DA023643) centered on decoding the tremendous heterogeneity among brain serotonergic neurons. Serotonergic neurons are broadly defined by their production of the major neurotransmitter serotonin (5-hydroxytryptamine, 5HT) and are implicated in human disorders ranging from psychiatric conditions of mind, mood, aggression and drug-seeking behaviors to disorders of cardiorespiratory homeostasis. Here, through a Revision (supplemental) Aim, we propose to delineate from the many different types of 5HT neurons, those most contributory to aggression in mice. Delineation of this key subset of 5HT neurons would be transformative, not only to our understanding of the neural circuitry underlying aggression modulation, but also toward identifying potential behaviorally selective therapeutic targets and/or diagnostics. These goals are central to OBSSR and NOT-OD-10-032. Enabling this scope-expansion of R21 DA023643 to include behavioral 5HT circuitry mapping are recent advances from my lab: (1) generation of a molecular framework that classifies murine 5HT neuron subtypes based on gene expression differences;(2) generation of the recombinase-based transgenic tools that provide in vivo genetic access to each of these identified 5HT neuron subclasses (original aim of R21 DA023643);and (3) development of new 'chemico-genetic'and 'exocyto-genetic'neuronal silencing transgenics, that when partnered with our cell subtype selective recombinase tools allow us to separately silence in the mouse each of the 5HT neuron subclasses and assay the impact on, and thus their role in modulating, aggression. This work is innovative, using novel behavioral genetic approaches, yet feasible because we have all necessary tools and expertise in place;further, it is of clinical importance - pathological aggression and its sequelae (such as higher rates of depression, anxiety, post-traumatic stress disorder and worse overall health functioning such as lower cognition and increased somatization) impair our society at all levels, from our youth to our elderly, and is heightened by pervasive factors such as alcohol and other drugs of abuse. Notice NOT-OD-10-032 Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications (R01, R15, R21, R21/R33, R37) through the NIH Basic Behavioral and Social Science Opportunity Network (OppNet). PUBLIC HEALTH RELEVANCE: Pathological aggression is a pervasive health problem with identifiable genetic, biological, environmental and treatment correlates. Underlying brain circuitry involves neurons that produce the major neurotransmitter serotonin, and clinical management commonly involves serotonin-related therapeutics - yet management strategies are hindered by the many other behavioral and physiological processes also modulated by serotonergic neurons. Here we propose cutting-edge molecular genetic studies in mice aimed at identifying the specific subset of serotonergic neurons most critical for aggression modulation;delineation of this subset would be transformative toward identifying behaviorally selective therapeutic targets and/or diagnostics.

[unreadable] DESCRIPTION (provided by applicant): A fundamental challenge in neuroscience and regenerative medicine is to understand how specific neuron subtypes arise in correct number, at defined times, and target to precise locations. Programs enacted to coordinate such events have begun to be defined, for example, through studies of early-stage spinal neural tube; in part due to physical and molecular accessibility, and relatively simple anatomy--newly born neurons typically move short distances radially, maintaining originating DV/AP relationships. Less clear is how such events are regulated in germinal zones that distribute neuron cohorts to distant and varied locations through tangential migration modes, and which typically do so at later developmental stages. Yet such germinal zones contribute substantially to neuron diversity in the brain and must do so in novel ways given their unique properties and the distinct milieu of late-gestation neural tissue. This proposal seeks to fill key aspects of this knowledge gap through studies of the hindbrain (lower) rhombic lip (LRL), a late-acting and essential germinal zone now accessible by molecular genetic means. Diverse and functionally critical brainstem cell types arise from the LRL, many subject to developmental and degenerative disorders. We provide evidence that (1) this diversity in produced cell types is reflected in a precisely defined molecular sub-regionalization of the LRL - the LRL is not homogeneous, but rather comprised of DV and AP gene expression microdomains predictive of progeny neuron identity; (2) the transcription factor Pax6 modulates aspects of this molecular sub-regionalization, ensuring that correct proportions of descendant lineages are produced; and (3) the archetypal ventralizing morphogen, Sonic hedgehog (Shh), is expressed in the dorsally situated hindbrain choroid plexus epithelium, a known dorsal organizing center and itself a LRL lineage. Such co-opting of previously excluded patterning molecules, such as Pax6 and Shh, may represent a general strategy by which a late-acting germinal zone may continue generating unique progenitor cell states and fates. Our goal is to two-fold: Extend our LRL fate map by linking highly resolved DV:AP coordinates to later-generated cell fates (Aim 1); place on this map the action of molecules which regulate this molecular organization and thus production of specific cell types; for example, we will explore novel roles for Pax6 and Shh (Aims 2 & 3). [unreadable] [unreadable] [unreadable]

Based on observations from brainstem tissue of SIDS cases, we hypothesize, as a program, that an important subset of SIDS result from medullary defects in serotonin (5-HT)-producing neurons and related neurotransmitter systems such as GABA. Evidence suggests that these defects arise during gestation and affect specific subtypes of, as opposed to all, 5-HT neurons. From this, we reason that SIDS is an embryonic developmental disorder affecting specific subtypes of 5-HT and/or GABA neurons of the medulla. Towards understanding the differential basis for SIDS, we propose experiments designed to decode the developmental and molecular origins of different neuron subtypes within the medullary 5-HT and GABA neural systems in the mouse, and determine the specific properties of these subtypes as relates to homeostatic control. These experiments are made possible via recent, powerful advances: 1) a developmental map of the mature brainstem 5-HT system that, for the first time, resolves the system into molecularly separable, and therefore genetically accessible, 5-HT neuron subtypes;2) the identification of embryonic genetic programs that are instructive for different GABAergic fates and which are likely employed in the developing medulla;and 3) tools with sufficient specificity to perturb the activity of (for example, "silence") select 5-HT or GABA subtypes in the living mouse. Using these tools, we will plot cellular functions and electrophysiological properties onto the developmental maps of medullary 5-HT and GABA neuron subtypes (Aims 1 and 3, respectively). Because our goal is to identify neuron subtypes most relevant pathophysiologically to SIDS, we will focus on 5-HT and GABA neuron subtypes of the medulla and their properties as relates to sensing acidosis and/or hypoxia (in collaboration with Project 4) and their functions as relates to control of breathing, heart rate, blood pressure and reflex apnea (in collaboration with Project 2). These are functions which, if impaired, might plausibly contribute to sudden death. Further, we propose investigating a possible mechanism for regulating 5-HT neuron production, our goal being to decipher the basis for the increased number of 5-HT neurons in SIDS cases. The ability to redefine medullary 5-HT and GABA neuron subtypes and their production based on a constellation of criteria - molecular, developmental, electrophysiological, and functional- is a major strength and innovation of this proposal and program.

[unreadable] DESCRIPTION (provided by applicant): Studies of neural circuit function and development would be advanced considerably by tools capable of "dialing down" or "turning-off" neurotransmission from select cells in the awake, freely behaving mouse. Especially powerful would be tools capable of effecting inducible and reversible suppression of neurotransmission, and which could do so with great cellular specificity - where the choice of cell type to be silenced is based on combinatorial codes of gene expression and thus is highly selective for a particular neuron subtype. Such a tool would allow causal relationships to be defined between a highly select neural circuit, gene expression (concurrent or antecedent) and animal behavior. Further, the features of inducibility and reversibility would allow silencing to be triggered during discrete developmental periods followed by recovery; from this it would become possible, for example, to reveal periods of circuit development during which select neuron activity may be critical for later circuit health - without which mature circuit function may be compromised or decline prematurely. Thus developmental windows of vulnerability for genetic or environmental insult could be identified. Here we propose developing and testing four genetic tools - four independent mouse strains - each designed to offer these capabilities, but each likely to differ in the kinetics and efficiency of neuron inactivation and recovery and thus in the types of circuits and behaviors most suitable for study. We will build upon a prerequisite and exciting set of tools already established in the lab: we will take component elements from a set of alleles, ones that we have recently shown able to visualize and silence a wide range of neuron subtypes in highly selective fashion, and now incorporate along with these proven elements other sequences that should allow for inducible and reversible silencing. By engineering these elements into single broadly applicable alleles, we simplify the experimental paradigm with respect to both time and expense. The innovation of the proposed work lies not in an individual genetic element but rather in how these elements will be used together - the resultant tools should have the potential to dramatically change neuroscience research through their use by many investigators to study the function and development of virtually any circuit in the mouse nervous system. PUBLIC HEALTH RELEVANCE A fundamental challenge of contemporary neuroscience is to define causal relationships between the activity of a given brain circuit and a particular animal behavior, including the developmental and molecular events that tie this relationship together. Towards meeting this challenge (one defined by this RFA-MH-08-060), we propose to develop tools for visualizing and manipulating the development and activity of discrete neural circuits (molecularly defined) in the awake, behaving mouse or in developing embryos otherwise undisturbed in utero. If successful, these reagents will have the potential to radically advance numerous areas of basic and translational neuroscience research; indeed, our hope is that these tools, generated by a single lab and R21 funding mechanism, will be leveraged, through their use by many neuroscientists, to advance studies of virtually any neural circuit in the mouse nervous system. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A fundamental challenge in neuroscience and regenerative medicine is to understand how specific neuron subtypes arise in correct number, at defined times, and target to precise locations. Programs enacted to coordinate such events have begun to be defined, for example, through studies of early-stage spinal neural tube;in part due to physical and molecular accessibility, and relatively simple anatomy--newly born neurons typically move short distances radially, maintaining originating DV/AP relationships. Less clear is how such events are regulated in germinal zones that distribute neuron cohorts to distant and varied locations through tangential migration modes, and which typically do so at later developmental stages. Yet such germinal zones contribute substantially to neuron diversity in the brain and must do so in novel ways given their unique properties and the distinct milieu of late-gestation neural tissue. This proposal seeks to fill key aspects of this knowledge gap through studies of the hindbrain (lower) rhombic lip (LRL), a late-acting and essential germinal zone now accessible by molecular genetic means. Diverse and functionally critical brainstem cell types arise from the LRL, many subject to developmental and degenerative disorders. We provide evidence that (1) this diversity in produced cell types is reflected in a precisely defined molecular sub-regionalization of the LRL - the LRL is not homogeneous, but rather comprised of DV and AP gene expression microdomains predictive of progeny neuron identity;(2) the transcription factor Pax6 modulates aspects of this molecular sub-regionalization, ensuring that correct proportions of descendant lineages are produced;and (3) the archetypal ventralizing morphogen, Sonic hedgehog (Shh), is expressed in the dorsally situated hindbrain choroid plexus epithelium, a known dorsal organizing center and itself a LRL lineage. Such co-opting of previously excluded patterning molecules, such as Pax6 and Shh, may represent a general strategy by which a late-acting germinal zone may continue generating unique progenitor cell states and fates. Our goal is to two-fold: Extend our LRL fate map by linking highly resolved DV:AP coordinates to later-generated cell fates (Aim 1);place on this map the action of molecules which regulate this molecular organization and thus production of specific cell types;for example, we will explore novel roles for Pax6 and Shh (Aims 2 &3).

Project IV seeks to identify molecular programs and gestational stages in mice that, if compromised, may cause homeostatic dysfunction, and, by extrapolation to humans, may underlie an increase in risk for the sudden infant death syndrome (SIDS) or other clinical disorders of homeostasis. Life- sustaining homeostatic responses to cardiorespiratory and thermal challenges are regulated by brain serotonergic (5-HT) neurons. Using novel mouse transgenics, we recently established that different homeostatic functions, for example respiratory control In response to CO2 elevation or body temperature control In response to cold, map to distinct ontogenetically defined subtypes of 5-HT neurons. This suggests that molecularly distinct 5-HT neurons mediate distinct functions. In addition, we now know that mice experience a window of heightened vulnerability to homeostatic stressors, spanning postnatal day (P)~5-12, reflecting that these homeostatic functions are developmentally regulated, and may be analogous to the critical period where human SIDS risk is elevated. We also know that male mice show a greater homeostatic sensitivity to 5-HT disruptions. In-line with the higher rate of SIDS in male Infants. Armed with these functional parameters - 5-HT neuron subtype, vulnerable postnatal stage, and gender - and our novel circuitry mapping tools, we propose to Identify molecular programs underlying these homeostatic specializations and vulnerabilities In mice. We hypothesize that critical molecular differences driving homeostatic specializations and their temporal development in 5-HT neurons stem from differences in gene expression and that they can be revealed through systematic comparison among transcriptomes generated from each of our Identified functional classes of medullary 5-HT neurons across postnatal windows Identified as especially vulnerable to homeostatic challenge and across gender (Aim 1). Further, we hypothesize that gestational exposures which elevate SIDS risk do so by perturbing the expression of critical homeostatic specializations, and that these perturbations Involve, at least in part, transcript alterations which are identifiable by comparative transcripfional profiling (Aim 2). Such gestational exposures may not only affect gene expression but also 5-HT neuron activity in the embryo, which in turn may affect the long-term development and postnatal function of homeostatic circuits. Using inducible genetic neuronal 'silencing' tools recently engineered In our lab, we will Identify, In mice, embryonic stages during which 5-HT neuron activity is most critical for development and function of circuits essential for postnatal homeostasis (Aim 3).

DESCRIPTION (provided by applicant): The proposed research brings cutting-edge genetic tools to bear on understanding the neural circuitry under- lying aggression and motivated behavior. Our recent progress reveals that these behaviors in mice are regulated by two molecularly distinct, small subsets of brain serotonergic (5-HT) neurons: one subset, ~3,000 neurons, uniquely defined among 5-HT neurons by expression of the D1 dopamine (DA) receptor (Drd1a gene), and the other, ~1,000 neurons, by the D2 DA receptor (Drd2 gene). These results combined with the enabling genetic tools, like a powerfully sharpened wedge, can now be used to break open and access the circuitry, cellular properties, and molecular pathways underlying these consequential behaviors. Here we propose applying this wedge in the form of four aims to answer: What cellular and molecular properties are unique to these behavior-critical 5-HT neurons? As suggested by receptor expression, are these 5-HT neurons responsive to DA - a neurochemical commonly associated with the reward system of the brain? Through what forebrain circuitry do these subtypes modulate aggression and motivation? Do these parameters change across the life span, perhaps bearing on human age-related propensity for impulsivity, aggression, and substance abuse? In Aim 1, we will identify functional properties of the Drd1a and Drd2 5-HT neuron subtypes by transcriptional profiling (RNA-seq) and electrophysiological recording. This work is enabled through novel genetic tools for neuron subtype-specific marking, suitable for neuron subtype sorting and molecular profiling, and for whole-cell recording. These same genetic tools not only offer access to the soma of a 5-HT neuron subtype, but also to axons and terminals, thus allowing precise identification of target brain regions under neuron-subtype control - the goal of Aim 2. Functional postsynaptic connections will also be explored, with our intersectional genetic marking tools conferring unprecedented resolution to classic tract- tracing techniques as well as to cutting-edge viral approaches that involve trans-synaptic tracers. Thus, Aim 2 will define, label, and allow for molecular characterization of aggression-relevant postsynaptic neurons downstream in these circuits. In Aim 3, we will explore more deeply the behavioral facets modulated by these two 5-HT neuron subtypes and if their contributions vary across life span. Similar subtype-specific silencing methods will be employed as in the foundational aggression studies, but now additional social behaviors and neurological functions will be queried. In Aim 4, we will use pharmacogenetics (DREADDs) to transiently silence each Drd 5-HT neuron subtype during childhood, asking if lasting changes occur that predispose to hyperaggression and altered social motivation in adulthood, as predicted by human studies that associate genetic predisposition via the 5-HT system, childhood stress, and adult pathological aggression. Our approaches are technically and conceptually innovative, and are foundational for discovering new, potentially behavior-selective, age-suitable therapeutics. Results compel a redefinition of 5-HT system organization.

DESCRIPTION (provided by applicant): Serotonin (5-HT)-producing neurons are recognized as key modulators of cocaine-seeking behavior. As cocaine-seeking behavior reflects the incentive motivational effect of stimuli paired with cocaine, 5-HT is thought to contribute to the formation of addiction-related memory that underlies relapse. Alteration of 5-HT commands is an emerging therapeutic direction for addiction treatment, with efforts focusing largely on receptor manipulations. Here we propose a novel, complementary approach that focuses presynaptically, on the 5-HT neurons themselves. Guided by recent research showing that cocaine seeking involves opposing activity of 5-HT at 5-HT2A and 5-HT2C receptors, which are key modulators of dopamine (DA) output, we will determine to what degree different subtypes of 5-HT neurons in mice differentially modulate the incentive motivational effects of stimuli paired with cocaine via the conditioned place preference procedure (Aim 1) and will explore their forebrain projection targets, especially as relates to postsynaptic 5-HT2A and 5-HT2C receptor expression (Aim 2). Our starting point is an emerging structure-function-connectivity map of the serotonergic neural system being assembled by the Dymecki lab, in which 5-HT neurons are classified by their expression of unique gene combinations and thus likely unique functionalities. They are further typed by their axonal target regions and by assessment of behavioral and physiological deficits following their selective silencing in vivo. Here we propose probing these 5-HT neuron subtypes for their role in addiction-related behavior, focusing first on 5-HT neuron subtypes that innervate brain regions implicated in enhancing or suppressing cocaine-seeking behavior. Three 5-HT neuron subtypes stand out: the r1-En1 5-HT neuron subtype, named by its origin in rhombomere (r) 1 and expression of the transcription factor Engrailed1; r2-Hoxa2 subtype, defined by its origin in r2 and expression of the transcription factor Hoxa2; and the Drd1a 5-HT neuron subtype, by expression of the type 1a DA receptor. Because the latter two show more restricted innervation profiles within the addiction-relevant mesolimbic system, perhaps suggestive of specialized roles in behavior modulation, these two will be explored first in this R21 application. Our approach of partnering molecular genetic techniques with a well-established behavioral assay in mice is both technically and conceptually innovative. The integration of these distinct disciplines is made possible only now through development of intersectional genetic tools that make molecularly distinct subtypes of 5-HT neurons apparent and accessible in mice for behavioral probing and genome-wide molecular profiling. Identifying the key 5-HT neuron subtypes involved and the nature of their effects on addiction-related behavior, along with having molecular genetic tools for their selective isolation and molecular probing is foundational for discovering new, possibly behaviorally-selective, therapeutic leads.

DESCRIPTION (provided by applicant): The sudden infant death syndrome (SIDS) is the sudden death of an infant that remains unexplained after a complete review of the history, autopsy, and death scene investigation; it is the leading cause of postneonatal infant mortality in the United States today. Based upon our findings to date, we hypothesize that SIDS is due to an underlying abnormality in the medullary homeostatic network that: 1) results in a failure of protective responses to life-threatening stressors during sleep in a critical developmental period; and 2) importantly involves serotonin (5-hydroxytryptamine, 5-HT), y-aminobutyric acid (GABA), and their potential interactions with other neurotransmitter systems and the signal transduction family 14-3-3. Initiated in 1998, our program brings together a highly committed group of 23 investigators and their trainees to address the potential role of the brain in SIDS. Located at 3 medical institutions across the country and the medical examiner's system in San Diego, CA, we bring to bear upon the SIDS problem outstanding expertise in multiple clinical and scientific disciplines in 3 integrated projects and 2 cores (Administrativ and Neuroanatomy). The 7 inter-related themes proposed for investigation with state-of-the-art methodologies in the next (fourth) cycle are: 1) gene expression profiles of the medullary homeostatic network in SIDS infants and animal models (Projects I and II; 2) the interaction of prenatal exposures (hypoxia and nicotine) with preexisting 5-HT dysfunction that potentially leads to homeostatic impairment in the postnatal period (Projects II and III); 3) the mechanism(s) of physiological derangements in protective homeostatic responses related to 5-HT and GABA, including autoresuscitation, arousal, and the laryngeal chemoreflex (Projects II and III); 4) the development and connectivity of subtypes of brainstem 5-HT neurons related to different homeostatic functions (Projects III); 5) a potential link between abnormalities in the caudal/rostral 5-HT domains of the brainstem and hippocampal targets in SIDS brains (Project I); 6) treatment of abnormalities in animal models with specific drugs (Project II); and 7) the development of future biomarkers of brainstem pathology in SIDS infants.

PROJECT SUMMARY/ABSTRACT Neuronal Firing Rate Homeostasis (FRH) describes the ability of neurons to maintain their average firing rates at precise set points in the long-term, even in the face of changing synaptic inputs. The stability provided by FRH enables learning and memory. Achieving FRH requires an FRH control circuit that is akin to a thermostat that compares desired to actual room temperatures or an autopilot that compares desired to actual trajectories. Identifying this control circuit will require direct measurement of FRH by perturbing firing rates and observing homeostatic recovery, which has rarely been done. A promising candidate FRH control circuit is the neuronal Activity-Regulated Gene (ARG) program, in which activity-dependent increases in calcium lead to transcription of genes whose mRNA levels remain elevated for many hours. The ARG program is a promising candidate because individual activity-regulated genes protect against seizures, and they also regulate homeostatic effector mechanisms like synaptic scaling that may mediate FRH. However, the molecules that comprise the FRH control circuit are unknown. We hypothesize that the ARG program is the core control mechanism of FRH. Our extensive preliminary data reinforce the importance of testing this hypothesis at genome-scale. The genome-scale approach is important not only because of the sheer number of ARGs but also because different ARGs appear to ?interpret? firing rate error in very different -- but as yet undefined -- ways. Relying on our extensive knowledge of the ARG program, we will evaluate its contribution to FRH. For ARG induction to be a core mechanism of FRH, it must be specified quantitatively by firing rates. We will therefore map the coupling of firing rates to ARG expression levels, by controlling firing rates optogenetically and detecting gene expression with RNA-Seq. We will also directly assess the functional contribution of ARG induction to FRH by perturbing firing rates pharmacologically in cortical neurons and observing homeostatic recovery using multi- electrode arrays, with or without perturbation of ARGs. Our approach is innovative because it is a genome- wide investigation of the activity-sensing control system for homeostasis rather than the far better-studied homeostatic effector mechanisms. Our work is significant because it will advance basic knowledge of the control circuit mediating FRH and produce tools for manipulating FRH that will help connect this control circuit to candidate homeostatic mechanisms.

Project Summary: A robust autoresuscitatory reflex (AR) is critical to newborn survival from birth. The transition to independent breathing and accommodation of breathing interruptions, apneas, that are common in neonates and infants, requires a coordinated cardiorespiratory response for recovery. 5-hydroxytryptamine (5- HT, serotonin) and the brainstem raphe cells that produce it, referred to as Pet1 neurons, organize and drive successful AR in newborn animals and humans. Unsuccessful AR is understood to be a major contributor to the sudden infant death syndrome (SIDS), where alterations in the brain 5-HTergic system have been described in ~half of human SIDS cases, including increased number of 5-HT neurons of differing morphology (smaller, simpler, perhaps immature), deficiencies in autoreceptor 5-HT1A binding, and decreased levels of 5- HT and tryptophan hydroxylase 2 (TPH2, the rate-limiting biosynthetic enzyme for 5-HT). We propose investigations to reveal in mice how aspects of this SIDS brain 5-HTergic phenotype develop prenatally. Our approach is informed by two recent findings. First, Pet1+ neurons in the raphe expressing high levels of 5-HT identity genes (e.g. Ddc, Vmat2, Gata3, Pet1) have been identified, smaller in size, with modest levels of autoreceptor 5-HT1A yet remarkably expressing little or no TPH2 and 5-HT. We call these novel cells para-5- HTergic neurons, signifying their partially shared molecular phenotype, shared location, and developmental emergence with 5-HT neurons. Second is the discovery of neurotransmitter switching, a noncanonical form of neuronal plasticity that occurs in response to stressors. Recent data support its role in shaping the 5-HTergic neuronal system, where stressors may drive some para-5-HT neurons to produce 5-HT as an adaptive response. Preliminary findings reveal that para-5-HT neurons derived from rhombomere (r) 4 densely and selectively innervate respiratory and arousal centers, and that gestational exposure to intermittent hypoxia results in an increased number of TPH2+ cells postnatally with as yet uncertain 5-HT levels. We propose that para-5-HT neurons are a pliant population that may be transformed when challenged prenatally by hypoxia to produce 5-HT in newborns as a compensatory mechanism to support AR. We hypothesize that in response to the major SIDS risk factor of prenatal hypoxia, certain para-5-HT neurons adaptively transform to produce 5-HT to rectify a 5-HTergic signaling imbalance that hinders the AR and, alternatively, that an insufficient transformation plays a critical role in SIDS. We will test this by exposing mice to intermittent hypoxia or normoxia during gestation, characterizing cellular and molecular phenotypes and querying neurotransmitter transformation (Aim 1); further, we will determine the effect of acute activation or inhibition of these r4-para-5-HT neurons on the AR in these mice (Aim 2), and we will examine phenotypic markers of para-5-HT neurons in human SIDS and control brain tissue (Aim 3). This novel, functionally defined, para-5-HT cell type and plasticity may be highly relevant to newborn viability.