Richard B. Levine - US grants

Insect metamorphosis is a time during which the nervous system is dramatically altered to allow the very different organism which emerges from the larva to behave in new ways appropriate to its new life situation. These pronounced changes in behavior require a striking reorganization of neuronal circuits and the properties of individual cells. Dr. Levine follows the changes in a mechanosensory reflex circuit that mediates a very different behavioral response at different life stages. Using innovative state-of-the-art techniques, he examines the mechanisms by which steroid and peptide hormones direct the formation of new synapses and modifies important cellular events which are reflected in the altered patterns of behavior. Recent findings have led to the conclusion that the adult nervous system is not rigid since adult neurons can undergo morphological changes. Dr. Levine's findings will enhance our understanding of the events underlying neuronal plasticity. Thus, his work holds promise of providing insights into how changes can occur in adult brains, especially with relationship to learning and memory and recovery of function.

DESCRIPTION: The development and maintenance of neuromuscular function depends upon reciprocal interactions between motoneurons and their target muscles. Motoneurons regulate the differentiation of muscle fibers and retrograde signals from the muscle fibers may be essential for the presynaptic specialization of motor terminals. Fundamental information about the mechanisms underlying these cellular interactions will contribute to our understanding of how proper neuromuscular function is maintained, and how it is compromised by congenital defects and disease. The insect nervous system has, for many years, provided a powerful model for investigating these mechanisms. During metamorphosis of the moth manduca sexta, larval leg motoneurons persist to innervate newly formed adult muscles following the degeneration of their larval targets. These motoneurons undergo a pronounced regression of their terminal aborizations and loss of presynaptic specializations, followed by regrowth and re-differentiation to allow innervation of the new adult muscles. The myoblasts that generate the adult muscles accumulate around the re-expanding motor terminals, then proliferate, fuse and differentiate. Accompanying these developmental events are precise changes in the titer of the steroid hormone, 20-hydroxyecdysone. The proposed experiments will examine the relative importance of reciprocal interactions between nerve and muscle, and hormonal cues, in regulating muscle development and presynaptic terminal remodeling. Several techniques, including electrophysiology, confocal and electron microscopy, activity-dependent staining of presynaptic terminals and immunolocalization of synaptic vesicle proteins will be used to compare presynaptic structure and function of the same motoneurons in the larva and adult, and the time course of motor terminal remodeling during metamorphosis. Confocal and electron microscopy will be used to determine the time course of myoblast accumulation and differentiation. in vivo manipulations, including denervation and the introduction of exogenous myoblasts, will be used to examine the role and specificity of motoneuron-derived signals in regulating the accumulation and differentiation of myoblasts. A nerve/muscle co-culture system will be used to further probe the mechanisms underlying these signals and to investigate the role of the ecdysteroid hormones in directing muscle development. Similarly, both in vivo and in vitro manipulations will be performed to examine the role of myoblasts and ecdysteroid hormones in regulating presynaptic differentiation. Basic knowledge derived from this well-defined model system will augment information being generated in other systems to provide a firm understanding of how motor systems develop and sustain normal function.

This proposal requests funds for the purchase of a computer- aided system for the reconstruction, measurement and statistical analysis of neuronal processes from sectioned or whole-mounted nervous tissue. The central goal of all the projects described is to understand how the detailed structure of neurons and their anatomical relationship?s with one another translate into the precise functioning of the nervous system. Five of the major users have projects that utilize various aspects of the insect nervous system as test subjects, a sixth investigates neural development in the amphibian nervous system.

Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS during postembryonic development. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential components of efforts to understand how the CNS is modified postembryonically to accommodate normal neural function and behavioral maturation, yet these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will take advantage of a well-characterized insect model system, where identified neurons undergo predictable phases of structural and functional modifications in response to defined steroid hormonal cues. The thoracic leg motoneurons of the moth, Manduca sexta, undergo dramatic dendritic and axonal regression and regrowth during metamorphosis to accommodate changes in their synaptic inputs an tar get muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20-HE ), acts directly on the motoneurons to regulate neurite outgrowth. The principal goal of the proposed experiments is to characterize the cellular consequences of steroid hormone action in vitro and relate them to the cellular mechanisms that are involved in the structural and functional remodeling of the leg motoneurons in vivo. In the first specific aim, video analysis will be employed to determine whether 20-HE enhances the growth of cultured leg motoneurons by stimulating branch formation, or promoting the retention of existing branches. Motoneurons placed in vitro just prior to the phase of neurite regression, rather than growth, will also be analyzed in comparison. Laser-scanning confocal microscopy images of dye-injected motoneurons will be used to examine similar questions in vivo. The second specific aim is to correlate steroid hormone effects on neurite growth and regression with modifications in the organization and stability of the cytoskeleton. Immunofluorescence, coupled with confocal microscopy, will be used to obtain high-resolution images of microtubules and actin filaments in cultured leg motoneurons that are undergoing 20-HE induced neurite growth or regression. Complementary experiments will employ electron microscopy to reveal cytoskeletal structures within identified motoneurons at crucial stages of development in vivo. The final aim is to test the hypothesis that 20-HE induced changes in voltage-gated Ca2+ and K+ channels, and consequent changes i Ca2+ influx, are essential elements of the intracellular pathway through which 20-HE regulates the remodeling of the leg motoneurons. We will determine whether changes in the levels of Ca2+ and k+ currents, that have already been described, are mediated by 20- HE and whether Ca2+-induced release of Ca2+ and K= currents, that have already been described, are mediated by 20-HE and whether Ca2+ -induced release of Ca2+ from internal stories is important for regulating structural remodeling. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.

The long range goal of the project is to understand the role of cellular interactions in the normal development and postembryonic remodelling of, neuromuscular systems. Interactions between motoneurons and their target muscles are a significant factor in the differentiation and maintenance of healthy muscle fibers, and target-derived information regulates the survival and morphology of motoneurons. Activity-dependent interactions are of particular importance during the later stages of development at the neuromuscular junction and during synaptic development within the CNS. Activity levels influence synaptic strength and specificity, and may play a crucial role during the postembryonic remodelling that is a normal part of the maturation of neuromuscular systems. In the proposed experiments, the remodelling of neuromuscular systems during insect metamorphosis will be employed as a model for examining the cellular mechanisms underlying nerve-muscle interactions. During metamorphosis in the moth, Manduca sexta, identified larval motoneurons survive the death of their target muscles and persist to innervate newly-generated muscles of the adult legs. The development of adult muscle is dependent upon the presence of the motoneurons. The availability of precise information about changes in the structure of identified motoneurons and in their expression of voltage-gated ion channels during metamorphosis, makes this an especially attractive model system. The initial aim is to identify the source of muscle precursors and to describe the differentiation of muscle fibers and neuromuscular synapses. The differentiation of voltage- and ligand-gated ion channels of muscle cells will be examined in detail. Experiments performed both in vivo and in vitro will reveal whether it is the proliferation or differentiation of muscle precursors that depends upon the motoneurons, and whether the neural influence is mediated by diffusible factors or requires cell contact. The specificity of the motoneuron requirement will be determined by manipulating identified motoneurons in vivo, and by varying the source and stage of motoneurons in neuron/muscle co-cultures. Further experiments will examine the role of neural activity in regulating the elaboration of motoneuron terminals and the differentiation of muscle cells.

It is proposed to develop an effective and efficient core facility that will provide routine technical assistance and well-maintained, up-to-date equipment for tissue culture studies associated with this Program Project. The instrumentation and physical facilities are already in place. The principal requirement to ensure optimal use of these resources is a Research Assistant to maintain the facility and perform basic duties in it.

The five participating research groups share a common interest in understanding the importance and mechanisms of intercellular and hormonal signals that regulate the postembryonic development and plasticity of neurons. Growing collaboration among these groups over the past eight years and increasing mutual focus on the mechanisms that control developmental plasticity within the nervous system have led to the emergence and continued success of this Program Project. Among the research areas represented by these laboratories are; the roles and mechanisms of short-distance and contact-mediated intercellular influences in neural development; hormonally regulated, postembryonic differentiation and remodeling of motor neurons and interneurons; ultrastructural and physiological aspects of synapse formation in d enveloping neural systems; the role of glial cells in the development of organized neuropil in the central nervous system; and molecular mechanisms of steroid hormone regulation of neuronal development. The participants will mount multidisciplinary, highly collaborative investigations of mechanisms underlying hormonal and trans-cellular regulation of the survival and development of nerve cells during postembryonic life. Specifically, the five component projects include: (1) molecular mechanisms of gene regulation by steroid hormones and characterization of steroid-sensitive genes; (2) morphological and biophysical development of CNS glial cells and their regulation by trans-cellular interactions; (3) steroid-hormonal regulation of development of identified motor neurons in primary cell culture; (4) molecular and genetic analysis of steroid hormone responsive genes and their roles in CNS development; and (5) cellular interactions and molecular mechanisms regulating the development of a primary olfactory center. All of the proposed studies are based on the use of invertebrate model systems that are economical, readily available, and experimentally favorable. Because they have been extensively studied by many investigators, including the participants in this program project, the insect preparations to be used in the proposed studies are especially well understood and favorable for these experiments. Specifically, Manduca offers excellent access to physiological and hormonal mechanisms, whereas Drosophila offers a powerful array of molecular and genetic approaches. The basic mechanisms of development have been conserved during evolution. We expect, therefore, that information obtained in these projects about neural and muscular development in insects will illuminate related phenomena in other animals including human beings.

The five participating research groups share a common interests in understanding the importance and mechanisms of intercellular and hormonal signals that regulate the post-embryonic development and plasticity of neurons. Growing collaboration among these groups over the past 13 years and increasing mutual focus on the mechanisms that control d4evelopmental plasticity within the nervous system have led to the emergence and continued success of this Program Project. Among the research areas represented by these laboratories are: the roles and mechanisms of short-distance and contact-mediate intercellular influences in neural development; hormonally regulated post-embryonic differentiation and remodeling of motor neurons and interneurons; ultrastructural and physiological aspects of synapse formation in developing neural systems; the role of glial cells in the development of organized neuropil in the central nervous system; and molecular mechanisms of steroid hormone regulation of neuronal development. The participants will mount multi-disciplinary, highly collaborative investigations of mechanisms underlying five component projects include: (1) the post-embryonic development of a highly-ordered region of the brain that is involved in associated learning; (2) analysis of glial- neuronal interactions in olfactory receptor neuron sorting and in formation of glomerular compartments during development of olfactory pathways; (3) steroid-hormonal regulation of development of identified motor neurons in vivo and in primary cell culture; (4) molecular and genetic analysis of steroid hormone responsive genes and their roles in CNS development; and (5) influence of male-specific olfactory sensory axons on the development of a specialized olfactory glomerulus and mechanisms of signaling by olfactory axons. All of the proposed studies are based on the use of invertebrate model systems that are economical readily Available, and experimentally favorable. Because they have been extensively studied by many investigators, including the participants in this program project, the insect preparations to be used in the proposed studies are especially well understood and favorable for these experiments. Specifically, Manduca and the cockroach offer excellent access to physiological and hormonal mechanisms, whereas Drosophila offers a powerful array of molecular and genetic approaches. The basic mechanisms of development have been conserved during evolution. We expect, therefore, that information obtained in these projects about neural and muscular development in insects will illuminate related phenomena in other animals including human beings.

DESCRIPTION: The Tissue Culture Facility provides the necessary equipment and facilities for culture experiments. Specifically, the facility is used for preparing insect primary neuron cultures and for maintaining hybridoma cell lines and insect cell lines. It is well-equipped for the services it provides. The facility is overseen by Dr. Richard Levine and is staffed by one full-time technician, Ms. Turner. She is responsible for maintaining the facility and all common-use reagent stocks. This facility will be used by all 5 PPG projects.

PROJECT 3: Hormonal influences on insect motoneurons during post- embryonic development. Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential for understanding how the CNS is modified post-embryonically to accommodate normal neural function, these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will exploit two well characterized insect model systems that offer complementary advances. During metamorphosis in the moth, Manduca, changes in their synaptic inputs and target muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20E) acts directly on the motoneurons of the appropriate stage to regulate neurite outgrowth and branching, as well as the levels of voltage- gated Ca2+ currents. The principal goals of the proposed experiments are to characterize the intracellular mechanisms of steroid hormone action and the relative influences of hormonal vs. inter-cellular interactions in regulating this remodeling. Whereas Manduca offers the advantages of large size for manipulation and electrophysiology and well-characterized endocrine titers. Drosophila, which undergoes similar motoneuron remodeling, offers powerful molecular-genetic approaches to exploring signaling pathways. In the first specific aim a new organ culture protocol and cell imaging system will be used to describe the dendritic modeling of identified flight motoneurons in Manduca and Drosophila in their natural context, in vivo, and to determine the roles of specific synaptic inputs and 20E. In the second specific aim, the intracellular 20E response pathway will be examined by describing the temporal expression patterns of ecdysteroid receptors and a key primary response key BRC in identified motoneurons as they are being remodeled. Steroid hormone regulation of key elements in the response pathway will be examined in primary cell culture. The requirement for specific primary response genes will be determined by examining appropriate Drosophila mutants, in collaboration with investigators of project 4. The third specific aim follows from our observation that 20 E affects both Ca2+ currents and the duration of internal Ca2+ increases, and that changes in Ca2+ current levels, in vivo, Ca2+ levels, Ca2+ release from internal stores, and the Ca2+-dependent enzyme, CAM-kinase II, in ecdysteroid-induced dendritic remodeling using voltage clamp, Ca2+ imaging and genetic approaches. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.

[unreadable] DESCRIPTION (provided by applicant): Normal brain function requires precise dendritic shape and electical characteristics. Neurons must acquire these properties during initial development and maintain them through life, except under circumstances where functional plasticity is required. The long-term objectives of this project are to understand the role of neuronal activity in regulating dendritic shape and distribution of voltage-gated ion channels. We have developed techniques for following the dendritic development of identified motoneurons during metamorphosis in the genetically-tractable model organism, Drosophila melanogaster, and demonstrated that activity levels influence dendritic development significantly. Neuronal activity and calcium-dependent transduction pathways can be manipulated precisely, both in vivo and in primary cell culture. Specific aims are: 1. To determine how the functional attributes of identified flight motoneurons are modified during metamorposis. Whole-cell current and voltage-clamp techniques will be employed. 2.To determine which forms of voltage-gated potassium and calcium channels are expressed at different stages, their distributions within the motoneuron and their regulation by steroid hormone. Channel expression will be measured by RT- PCR, and distribution determined by forcing the expression of channels tagged with a tetrad-cystiene motif. 3. Characterize the acitivity-dependent regulation of dendritic development. The activity patterns of identified motoneurons will be increased or decreased by driving the expression of modified ion channels and the effect determined with both in vivo and in vitro measurements of growth and function. 4. Determine the role of calcium-dependent signalling pathways. The source and nature of activity-dependent calcium influx will be determined using genetically-encoded calcium indicators, the highly conserved nature of neuronal ion channels and the molecular pathways that regulate developmental processes suggests that basic information derived from this model system will provide insights into human health. Relevance to public health: Normal brain function requires that neurons develop a precise dendritic structure and a defined array of electrical characteristics. Stroke and diseases, such as epilepsy, can result in improper maintenance of these properties. The proposed research seeks to characterize mechanisms that ensure proper development and maintenance of these characteristics. [unreadable] [unreadable]