Darcy B. Kelley - US grants

This proposal examines the characteristics, ontogeny and mechanisms of androgen-induced laryngeal proliferation. Our focus is on muscle and its androgen receptors. Experimental approaches include tritiated thymidine autoradiography androgen receptor assays, electron microscopy, steroid autoradiography and tissue culture. We will induce proliferation in the larynx of juveniles and characterize the temporal parameters of the response. Mitogenesis in different cell types (e.g., myoblasts, fibroblasts) will be examined at the light and electron microscopic level. The ontogeny of androgen-induced proliferation will be studied and related to sexual dimorphism and reproductive physiology at puberty. We will measure androgen receptors in laryngeal muscle and analyze receptor affinity and specificity in males and females at different ontogenetic states and in response to endocrine stimulation. Relations between androgen receptors and proliferation in myoblasts and fibroblasts will be examined in vitro. The long term objective of our research is to uncover mechanisms whereby hormones regulate sexual differentiation of neuroeffectors for reproductive behavior. We believe that a focus on the motor neuron-muscle unit will provide powerful advantages in such analyses. Results of these studies should shed light on steroid modulation of myogenesis and regeneration and on endocrine complications of neuromuscular disease.

The overall objective of this project is to determine how hormones interact with central nervous system effectors for reproductive behaviors. Our experimental approach combines techniques from behavioral neuroendocrinology, neuroanatomy, developmental neurobiology and neurophysiology. The focus of the present proposal is on the neural and hormonal control of vocal behaviors in the South African clawed frog, Xenopus laevis. This vertebrate experimental system offers sterotyped, sexually dimorphic reproductive behaviors under strict control by specific gonadal steroids. In the first set of experiments, we will investigate the hormonal control of two sex-specific reproductive vocal patterns: male mate calling and female ticking. The relative contributions of genetic and hormonal sex to the differentiation of behavioral repertoires will be determined. The existence of a "sensitive period" for hormone effect on behavior will be explored. In a second set of studies, we will map the central nervous system pathways for vocal control. The effects of sex-specific vocalizations on the reproductive behaviors and neuroendocrine state of males and females will be studied. The efferent vocal pathway and the afferent auditory system will be mapped anatomically. We will focus on intersections of limbic diencephalic projections with the calling pathways as these may provide the neuroanatomical bases for behavioral coordination of copulatory acts with vocalizations. In the final series of experiments, we will examine how sex hormones affect neural systems for reproductive behaviors. The activity and sensitivity of a sex hormone-concentrating midbrain auditory nucleus will be examined under different endocrine conditions. The sensitivity to sex hormones of neuroeffectors for reproductive behaviors will be examined in developing and adult stages. The distribution of steroid-sensitive CNS cells will be compared to the locations of cells and fibers containing the behaviorally important neuropeptiode, luteinizing hormone releasing hormone. The work proposed here forms a basis for understanding effects of hormones on behaviorally important neurons. Basic principles of endocrine-neural interaction underly future research in such clinically relevant problem areas as; the deermination of gender identity, the actions of oral contraceptives and hormonal control of fertility.

The goal of this research is an understanding of how hormones, particularly the steroids - androgens and estrogens, control the sexually specific development of target neurons and muscle to produce the mature male or female phenotype. These questions are central to an understanding of male- and female-typical clinical disorders. Hormone-directed cell development underlies malignancies of prostate and breast, disorders of secondary sexual differentiation such as androgen-insensitivity and pseudohemaphodism and at least some aspects of the establishment of gender identity. This proposal is focussed on the control of cell type in laryngeal muscle fibers and at the laryngeal neuromuscular junction of Xenopus laevis. This experimental preparation provides an excellent model system in which to study the cellular and molecular bases of secondary sexual differentiation. The development of the vocal organ, or larynx, is highly sexually dimorphic as a result of sex differences in the endocrine milieu. The isolated larynx can produce actual vocalizations permitting identification of male- and female-specific electrophysiological and muscular characteristics which underlie the different vocal repertoires of adult males and females. These differences include a sex difference in synaptic efficacy - weak synapses in males versus strong synapses in female - and in muscle fiber type -rapidly contracting fibers in males and slowly contracting fibers in females. The control of synaptic strength and the ability of synapses to increase in efficacy with use will be examined in males and female larynges using electrophysiological and dye imaging methods. Calcium entry into the presynaptic terminal will be followed using calcium-sensitive, fluorescent dyes; mechanisms underlying sex differences - including sex differences in ion channels and in other aspects of transmitter release- will be examined. The developmental origins of sex differences in synaptic efficacy will be explored. Preliminary results strongly suggest that sex differences in synaptic strength are regulated by exposure to estrogen. The way in which estrogen acts on the laryngeal synapse to promote neurotransmitter release will be studied and cellular and molecular mechanisms underlying estrogen action determined using hormone ablation and restoration, immunocytochemistry and in situ hybridization. A laryngeal-specific, androgen-regulated myosin heavy chain gene (LM) has been identified as a strong molecular candidate for the regulation of fiber type in the larynx. The role played by LM in fiber type determination will be examined in vivo and in vitro using molecular and histological approaches. Preliminary results suggest that LM is an embryonic myosin heavy chain (MHC) isoform. The expression of LM and of other Xenopus MHC's will be examined in tissue extracts, whole mounts and sections using hybridization techniques. The of LM and of other MHC's in laryngeal muscle by androgen and thyroid hormone will be explored in vivo and in vitro using endocrine ablation and replacement. The lineage of myoblasts giving rise to laryngeal fibers will be examined and the role of LM-expressing myoblasts in fiber type switching examined.

Our goal is an understanding of the cellular and molecular mechanisms responsible for central nervous system sexual differentiation. The present proposal explores the contribution of the masculinized periphery - muscle and neuromuscular junction - to this process. Our experimental system is the laryngeal muscle of Xenopus laevis, a sexually dimorphic, androgen-target muscle innervated by sexually dimorphic, androgen-target motor neurons. We have established that the marked sex difference in muscle fiber number of the adult is due to androgen-induced myogenesis during a limited developmental period. The goal of the proposed experiments is to determine the cellular basis of this process, clarify the role and site of action of androgenic steroids and determine whether the process of masculinization includes sexual differentiation of the neuromuscular junction. We will examine myoblast induction, survival and differentiation during male and female development in vivo and in vitro. Androgen receptor expression will be measured using binding assays and steroid autoradiography. Cell types will be identified from electron micrographs and by antibody and in situ hybridization labeling. The role of innervation in myoblast sexual differentiation will be studied by blocking muscle activity and by nerve section. We will characterize the development of synaptic innervation in males and females morphologically and examine acetylcholine receptor distribution. We believe that the sexual differentiation of motor neurons is regulated, at least in part, by masculinization of the periphery. Androgens can effect the development of sexual dimorphisms by gaining access to certain key developmental processes -- cell specification, survival, differentiation, and cell to cell contacts. We plan to determine how steroid hormones exert these effects and why some are limited to certain key developmental stages or "critical" periods. These questions are key to unravelling how the brain develops and the neural and endocrine differences that underlie the increased vulnerability of males to developmental abnormalities including some neuromuscular diseases, aphasias and affective disorders.

The overall goal of this research is to understand the mechanisms by which gonadal steroid hormones influence the development and adult function of neurons that participate in behavior. The effects of steroids on behavior are believed to be exerted by action on the central nervous system. There are marked differences in the central nervous system of males and females. Among the most striking of these are examples of differences in the number of motor neurons that innervate effector muscles for behavioral expression. These differences correlate with behavioral differences, but it is not clear how sexually dimorphic morphology contributes to sexually dimorphic function. Of considerable interest are the developmental processes that give rise to morphological differences. We do not yet know how sex differences arise in the neuroeffector systems that produce sexually differentiated behavior. This research contains experimental approaches to determine when and how sex differences in the motor neurons that produce sexually dimorphic behavior develop in the clawed frog, Xenopus laevis.

The aim of this research is an understanding of the cellular and molecular mechanisms that govern the sexual differentiation of the brain. In particular, we are investigating how male-specific patterns of secretion of gonadal androgens regulate the proliferation, survival, synaptic connectivity and function of target muscle and motor neurons. We have exploited a highly sexually dimorphic system--the neurons and muscles that control vocalizations in X. laevis--to identify key events in the development of functional sex differences. Our specific aims now are to further understand how androgen secretion controls sexual differentiation at the cellular level and how a set of molecules--the androgen receptor, gap junction proteins, myosin isoforms-- participate in this ontogenetic process. We will examine the development of sexually dimorphic muscle fiber types in larynx using histochemistry and immunocytochemistry. Myosin isoforms will be separated biochemically; we will determine when sex typical expression first diverges. We will investigate the molecular basis and ontogenetic history of dye coupling of muscle fibers in adult females. We plan to examine sex differences in acetylcholine release and number of synaptic terminals at the laryngeal neuromuscular junction. The origins of sex differences in laryngeal motor neuron number and sexually dimorphic CNS connectivity will be explored. We will measure androgen levels and receptor expression in brain just after the gonads differentiate. Using endocrine application and ablation, the critical periods for hormone action on key cellular and molecular processes will be established. The relative contributions of direct action on muscle and nerve targets versus indirect, synaptically mediated, actions will be assessed. Androgen secretion during development controls brain sexual differentiation in most vertebrates. Disorders in endocrine function or in androgen receptor proteins are associated with clinical dysfunction including pseudohermaphroditism, testicular feminization, neuroendocrine abnormalities and alterations in gender identity. An understanding of basic processes affected by steroid hormones in nerve and muscle can be derived from study in animal models and will provide useful insights into underlying cellular and molecular mechanisms of human disorders.

The proposed research continues studies of the origins, circulation and fate of membranes and of membrane-delimited compartments within frog retinal photoreceptors and other cells of the nervous system. The investigations will be based principally on electron microscopic cytochemistry, immunocytochemistry and autoradiography with supportive fluorescence microscopic, cell fractionation, physiological and biochemical determinations. Most of the work will concern two related facets of membrane circulation: 1. Studies focused on the sorting of membrane proteins to opposite poles of the photoreceptor (the photoreceptive outer segment and the presynaptic terminal) will aim at testing aspects of a model which proposes that sorting begins at the endoplasmic reticulum and continues from distinctive subregions of trans Golgi systems. This work will include studies of the normal functional organization of the Golgi apparatus and will also investigate the impact of agents expected to perturb transport and Golgi functioning in different ways and at different steps. 2. Studies on the cycling and fate of synaptic vesicle membrane in the photoreceptor terminals will concern particularly the influence of weak bases on these processes. (The bases raise intracompartmental pHs in the terminals, and engender the accumulation of structures with the properties of recycling intermediates--relatively large compartments of endocytic origin, which can later give rise to synaptic vesicles.) The details of processes by which recycled synaptic vesicles arise will be investigated. A principal aim will be to determine the involvement of intracompartmental pH and of osmotic phenomena in governing sorting and cycling in the photoreceptor. Participation of lysosome-related bodies in the turnover of synaptic vesicles will also be examined. Collaborative work will continue on identifying genetic mutants with altered peroxisomes. This work is aimed at obtaining material with which to investigate the likelihood that peroxisomes are important in the turnover of membrane components. Disease relatedness: Alterations in membrane cycling and in the turnover of membrane molecules are involved in many disorders affecting the retina and other nervous tissue. (Some of the studies will be directly on cell lines from individuals with such disorders.)

DESCRIPTION (provided by applicant): Continued support is requested for a training grant in neurobiology and behavior, which is about to enter its ninth year of funding. This grant supports students during their first two years in a degree-granting program, the Doctoral Program in Neurobiology and Behavior at Columbia University, which provides integrated academic and research training leading to a Ph.D. degree in neuroscience. The nervous system is the most complex of all tissues, and understanding its biology has required the combined forces of several areas of modern science, including cell biology, biochemistry, developmental biology, molecular biology, pharmacology, physiology, and psychology. The educational requirements of this field can be difficult to meet if constrained by long-established requirements of the traditional academic departments. Neuroscience faculty from throughout the university therefore joined together ten years ago to establish a university-wide, multidisciplinary training program to meet the educational needs of all predoctoral neuroscience students at the university. The 75 faculty members include many leaders in various areas of neuroscience, whose research interests span the range from molecules to cognition. This broadly based program provides coherent training via a unified admissions process, curriculum, and a comprehensive set of research opportunities. Trainees will have access to the facilities and resources of the Center for Neurobiology and Behavior and the Departments of Anatomy and Cell Biology, Biochemistry and Molecular Biophysics, Biological Sciences, Genetics and Development, Neurology, Pathology, Pharmacology, Psychiatry, and Physiology and Cellular Biophysics, Psychology, and Statistics. Major areas of strength in training expertise include both basic and translational areas of neuroscience, including: Stem Cell Biology, Cell Differentiation and Migration, Axon Pathfinding and Synaptogenesis, Biophysics/Ion Channels/Transporters, Synapses and Circuits, Neurobiology of Learning and Memory, Cognitive/Systems Neuroscience, Animal Models of Psychiatric Disorders, Neural Degeneration and Repair, Brain Imaging and Theoretical Neuroscience. There are currently a total of 69 students in the program. Support is requested for five students during the first two years of training (10 total/year), to rise to 12 (6x2) by the third year.

DESCRIPTION (provided by applicant): Common to all vocal communication systems is matching between hearing and utterance. The match can be achieved via learning (as in song birds or humans) or be innate or largely unlearned (as in frogs or non-human primates), but in both cases strong selective pressures have shaped the ability to produce appropriate vocal responses to specific acoustic signals. How this task is accomplished by the neural networks that produce vocal responses is not well understood in part because of the complexity of most experimental systems. The long-term goal of this research is to understand neural mechanisms for auditory/vocal communication through exploration of a well-established model system: vocal communication in the South African clawed frog, Xenopus laevis. Many aspects of the neural circuitry underlying vocal production have been worked out and a reduced preparation - the isolated brain- can be induced to produce fictive calling. We are particularly interested in understanding the role of a forebrain nucleus, the ventral striatum (VST) which - uniquely in the Xenopus vocal circuit - receives auditory information and projects directly to the major hindbrain afferent of vocal motor neurons, nucleus DTAM. The central hypothesis of this research program is that the VST participates in selecting an appropriate vocal pattern in response to specific acoustic input. To investigate this hypothesis we first need additional information about how the VST functions as a pre-motor and an auditory nucleus. Production of vocal signals is controlled by a set of neuromodulators whose effects depend on endocrine state. Thus an additional goal of this phase of the research is to understand how neuroendocrine factors such as gonadotropin and neuromodulators such as serotonin affect the function of the VST in the vocal circuit. Our experimental approaches combine in vitro and in vivo methods. We use the isolated brain preparation we have recently developed to understand the participation of various components of the auditory/vocal neural circuit in the vocal response;cellular and molecular approaches are combined with stimulation and electrophysiological recording. We will test proposed mechanisms in vivo to determine to what extent whether they actually participate in the endogenous control of vocal behavior in the animal. The ability of forebrain to influence vocal output could derive from an ancient set of neural connections that have been elaborated in some vertebrates. If so, the forebrain/hindbrain, auditory/vocal system in Xenopus laevis should provide very interesting insights into the ways in which neural networks for communication can function as well as suggesting ways in which communication disorders can be treated. PUBLIC HEALTH RELEVANCE Several human speech disorders are attributable to impaired function of auditory/vocal linkages. Deafening before language acquisition has profound effects on development and communicative skills [64]. When prelingually deaf adults receive cochlear implants, the quality of the speech production improves along with speech perception [65]. Articulation disorders, which are commonly regarded as motor in origin, such a stuttering and aphasia, can improve, sometimes quite dramatically, by auditory cues delivered in a social context such as choral speaking [66] and singing [67]. The effects of choral speaking, in particular, have been proposed to be mediated by the "mirror neuron" system that supports imitation in general and vocal imitation in particular. form the basis for "mirror neuron" systems proposed to function in human speech and learned bird song. Auditory/vocal linkages in forebrain may, in fact, arise from a set of evolutionarily ancient mechanisms that link hearing to utterance. If so, understanding auditory/vocal matching in Xenopus could provide a set of candidate neural mechanisms whose prevalence and utility can then be investigated more widely.

Amphibians (frogs and salamanders) are key indicator species for environmental change; many are threatened by habitat loss, rising sea levels and changing temperatures as they are “cold-blooded” and do not regulate body temperature. Some species, however, are resilient in the face of climate change both in physiology (e.g., temperature regulation), developmental requirements, and changes in behavior produced by the activity of nerve cells in the brain and spinal cord. African clawed frogs (Xenopus), though they live in fresh-water throughout life, can sequester in small chambers underground for very long periods when their environment becomes dry and hot. Xenopus used these resilience strategies to survive global extinction events. Spanish ribbed newts (Pleurodeles) can regenerate their entire nervous system, even as adults. To understand why these particular amphibians are so hardy, we need to find out how particular parts of their bodies work under stressful conditions. This project aims to develop “viral vectors”, non-infectious viruses that can be delivered to, and manipulate, genes in different parts of the body. These vectors can help test ideas about, for example, which parts of the brain are involved in resilience in frogs and how newts and salamanders regenerate whole parts of the body when they are injured. Also, the process of finding viruses that can infect amphibians will help investigators using other species such as birds and may reveal new ideas about how the ability of a virus to infect a different host species evolves, leaping from bats, for example, to humans. The project also includes training of undergraduate and graduate students, exposing them to international team science, as well as conferences and workshops, and sharing of protocols and non-infectious viruses on public databases to enable similar research by other investigators.

Viruses - natural multigene expression and delivery vehicles - evolved to target different species and tissues. Engineering Adeno-Associated Viruses (AAVs) for cold-blooded vertebrates (semi-aquatic or aquatic amphibians) is the focus of this EDGE project. Recombinant AAVs production enables a directed evolution approach for high-throughput selection and screening in two amphibians: the anuran Xenopus and the newt Pleurodeles. This research characterizes the blood brain barrier in both species to identify whether – or at what developmental stage – it forms. Leveraging the NSF-supported CLOVER Center at CalTech, researchers intravenously deliver an AAV serotype that transfects both species; they then harvest the animals’ central nervous system to produce, sequence, and bioinformatically analyze the resulting variants through two rounds of screening. Because of limits in the carrying capacity of AAVs, the project is developing transgenic cre lines that express specifically in neurons for both species. Using AAVs carrying floxed-CRISPR constructs and validated gRNAs, investigators knock out two native genes – rhodopsin and tyrosinase – in the eye via intraorbital delivery. Knocks outs are verified immunohistochemically using validated antibodies. AAVs are shared at cost with collaborators and deposited in Addgene. Results are shared via a US-based virtual conference, a hands-on US workshop, and an international conference. Protocols and validated results are rendered available to the broader research community via organism-based websites (e.g., Xenbase). All data and protocols are deposited in a publicly available data base and archived at Columbia University.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.