Lynn T. Landmesser - US grants

The long term goal is to understand the mechanisms by which chick spinal cord motoneurons form behaviorally appropriate circuits during neural development. Such basic mechanisms play a role in nerve and spinal cord regeneration, and in plastic changes of the nervous system in response to injury. The studies are also relevant to developmental defects of both nerve and muscle. Embryonic surgery and vital staining of identified neurons with fluorescent probes will allow the dynamic behavior of the migratory muscle precursor cells and neuronal growth cones to be documented in cultured slice preparations using time lapse cinematography and video-microscopy. The molecular nature of factors that affect patterned neuronal outgrowth will be assessed by the application of poly and monoclonal antibodies to such slice preparation (i.e. antibodies to NCAM, laminin, etc.). Both polyclonal and monoclonal antibodies will be generated to limb and spinal cord at the stages of initial axonal outgrowth, in an effort to obtain antibodies that block specific growth cone decisions. Finally, the glycogen depletion technique will be used to probe the role of motoneuron cell death and functional activity in refining motor projections within muscles, including the matching of fast and slow motoneurons and muscle fibers.

The long-term goal of the proposed research is to understand how functionally appropriate synapses are established during development. The aspects of synapse formation to be examined are how neurons acquire their repertoire of neurotransmitters and how target tissues acquire their complement of transmitter receptors and effector proteins. These questions will be addressed in the sympathetic nervous system because (1) the transmitters and receptors are well characterized and tools exist to study them, (2) culture techniques permit dissection of mechanism in a reduced system and (3) development which occurs postnatally is accessible to manipulation in vivo. Previous studies revealed that during normal development, a population of sympathetic neurons changes their transmitter phenotype, from noradrenergic to cholinergic. This switch is retrogradely specified by a soluble factor(s) produced by their target tissue, sweat glands. While innervation is not required for sweat glands to express muscarinic receptors, the appearance of cholinergic function in sweat gland neurons is required for the glands to develop secretory responsiveness. We now plan to determine whether sympathetic neurons, in addition to those that innervate sweat glands, undergo a transmitter switch and if so whether the target induces it. The role of target tissues in determining neuronal phenotype will be examined in several other paradigms. Tabby mutant mice lacking sweat glands will be used to determine if target tissues are required to guide sympathetic axons. Mice in which the CDF/LIF gene has been "knocked out" will be studied to establish whether CDF/LIF plays a role in the transmitter switch of sweat gland neurons. Transplantation experiments will be used to determine whether sympathetic target tissues transneuronally specify the neuropeptide content of their preganglionic innervation. Retrograde tracing will be combined with axotomy to assess the stability of target-specified transmitter properties in sweat gland neurons. We will also determine which aspects of target tissue development are regulated by innervation. Preliminary evidence suggests that innervation regulates cholinergic differentiation factor production by sweat glands. We will ascertain whether catecholamines are responsible. Other receptors in sweat glands will be examined to determine if their expression is controlled by innervation. The expression of candidate effector proteins required for responsiveness and regulated by cholinergic innervation will be studied. Finally, we will determine whether a critical period exists for target-induced changes in transmitter properties. These studies will elucidate the cellular and molecular mechanisms responsible for normal synaptogenesis and provide insight into where this process may be disrupted during abnormal development.

DESCRIPTION (provided by applicant): Understanding normal brain development and function and how it is altered by disease, injury, or environmental factors is one of the most exciting frontiers remaining in biomedical science today. New knowledge and tools acquired over the past decade offer hope for the development of new therapies for neurodevelopmental disorders, psychiatric illnesses, spinal cord injury, stroke, and neurodegenerative diseases. However, to effectively apply basic science knowledge to address these neural disorders requires the training of a new generation of neuroscientists. The goal of this training program is to provide five trainees in the first two years of Ph.D. training with a deep understanding of nervous system function and dysfunction at multiple levels of organization (molecular, cellular, circuit, behavior) and with the ability to apply diverse approaches (molecular/genetic, physiology, imaging) to understand how the nervous system develops, functions, and responds to injury or disease. This will be achieved by a program of formal course work and laboratory rotations with a highly interactive group of trainers whose expertise spans a broad range of neuroscience, in addition to active, continuous self-learning though participation in journal clubs, outside seminars, and other interactive forums. The program is aimed at equipping the trainees with the skills needed to identify and solve important problems throughout their careers as independent scientists. RELEVANCE: The training provided by this program will enable a new generation of neuroscientists to apply their knowledge of basic neuroscience mechanisms to develop therapies for neurodevelopmental disorders, psychiatric illnesses, spinal cord injury, stoke, and neurodegenerative disorders.

[unreadable] DESCRIPTION (provided by applicant): The proposed research is designed to determine the role that rhythmic spontaneous electrical activity plays n the development of spinal motor circuits required for normal locomotion. In-ovo drug induced alterations in the frequency of such activity in chick embryos from stages 20-23 was recently shown to alter the expression of specific adhesion/guidance molecules such as PSA-NCAM, EphA4, and CRYPD, to cause alterations in axon fasciculation, and to produce motor axon pathfinding errors. The mechanisms underlying these alterations, including changes in the bursting frequency versus signaling via specific transmitter systems (ACh, GABA etc.), will be elucidated by combining in-ovo application of specific receptor antagonists with exogenous electrical stimulation at different frequencies. Cultured vibratome slices will be used to further define the intracellular signaling cascades that link rhythmic activity to the expression of specific molecules required for proper circuit formation. Ca2+ imaging in these slices will determine how soon newly generated motoneurons and interneurons become incorporated into functional circuits and thus subject to activity dependent regulation. The specific parameters of the activity used and the downstream signaling pathways that are activated will be determined. Attempts to restore motor function after spinal cord injury by either the regeneration of descending inputs or by the exogenous activation of existing circuits will benefit by the more complete understanding of the effects of activity on the formation and maintenance of cord circuits that these studies will provide. The proposed research should be relevant to causing the differentiation of specific motor and interneuron subtypes from stem cells. It will also identify defects in human fetal spinal cord development that could result from maternal ingestion of nicotine as well as other medically prescribed drugs that alter cholinergic or GABAergic transmission. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The proposed research is designed to elucidate the cellular and molecular mechanisms underlying the recently identified roles of the different isoforms of NCAM in bringing about the structural and functional maturation of the neuromuscular junction required for normal motor function. It is specifically designed to define the role of the 140 and/or 120 kD isoform of NCAM in the down regulation of the immature, brefeldin A and L-type Ca+2 channel sensitive, immature vesicle cycling/release mechanism from the axon. It will also explore how the 180 kD isoform results in the appropriate localization of presynaptic molecules required for mature, effective transmission. Finally, it will elucidate the role of the highly conserved C-terminal domain of NCAM, which in concert with myosin light chain kinase and PCKe, has been shown by this work to be required to sustain effective transmission at adult synapses in response to repetitive stimulation. These goals will be achieved by electrical recordings and FM1-43 imaging of synapse formation in motoneuron myotube cultures from wild type mice or those lacking specific NCAM isoforms. Intracellular signaling and protein-protein interactions in isolated adult nerve-muscle preparations will be investigated by the introduction of specific blocking peptides into the presynaptic terminal. The formation of effective synapses is essential for normal neural function in both the peripheral and central nervous system. Mutant mice lacking NCAM, while viable and fertile, display motor defects as well as alterations in learning and memory. Similar defects are likely to occur in humans with mutations in the NCAM gene. In addition, these studies will provide insight into steps in presynaptic maturation and vesicle cycling mechanisms that are required for junctions to function at high but physiological repetition rates.

DESCRIPTION (provided by applicant): The goal of the proposed research is to optimize the ability to control with great spatial and temporal precision the levels of cAMP in specific subsets of vertebrate neurons by using a light-activated adenylyl cyclase, PAC. By placing this cyclase under the control of cell specific promoters it should be possible to control cAMP levels non-invasively by light in dissociated neurons, slice cultures and in intact developing embryos in vivo. Using FRET based measurements via a cAMP reporter, the cAMP transients produced by brief flashes of 455nm light in explant cultures of embryonic chick and mouse motoneurons electroporated with PAC will be characterized in somas and growth cones and compared to those produced by endogenous spontaneous electrical bursts in the same cells. Such transients will also be characterized in more intact cord-limb bud slice cultures. The effect of cAMP transients on the ability of motoneurons to respond to inhibitory and attractive guidance cues will be tested in dissociated neurons. Their effect on motoneuron dorso-ventral pathfinding will be tested in cord-limb bud chick and mouse slice cultures and in intact developing chick embryos in ovo. PAC will be placed under the motoneuron-specific promoter Hb9 to determine if it can selectively drive cAMP transients in motoneurons. Since intracellular cAMP has been shown to modulate how neurons interpret attractive and inhibitory growth/guidance cues, different patterns and frequencies of light activation of PAC will be used to determine if these can influence the response of cultured rodent DRG neurons to inhibitory signals. Based on a positive outcome, light activation of PAC in adult rodent DRG cells in vivo will be carried out to determine if this can enhance the regeneration of either the peripheral or central processes of transected DRGs. Given that cAMP modulates a vast array of important processes in the nervous system, the information gained from this study should facilitate the application of light-activated adenylate cyclase to control cAMP levels non-invasively in a variety of circumstances in intact organisms with widespread basic and translational applications, including enhancing the regeneration of axons in the CNS.