Kenneth J. Muller - US grants

Individual neurons in the leech can regenerate severed axons to reconnect with particular neurons that are their normal synaptic targets. Repair occurs in stages, from initial sprouting of the injured axon and growth that may be along selected pathways to recognition of the target, synapse formation, and cessation of growth. The proposed project addresses the questions: (1) what cellular interactions and capabilities control sprouting, (2) which cells and extracellular elements along the pathway might stimulate or direct growth, (3) are inappropriate as well as appropriate contacts made, (4) how do the new synapses compare with the old in distribution and function, (5) why do axons stop growing, and (6) what features of axon growth and synapse formation during development are revived during regeneration? The methodology will include intracellular and extracellular electrophysiological recording and intracellular injection of markers for subsequent light and electron microscopic examination. Growth of axons, filled with fluorescent markers, will be tracked in living preparations, in some cases maintained in culture medium. Properties of pathways and surfaces of particular cells will be examined, using electron microscopy, with cell-specific ligands including monoclonal antibodies and with freeze-fracture. Single cells can be killed with intracellular injection of proteases or focally lesioned using dyes that produce photodynamic damage. The morphology of certain cells and the complex patterns of normal and regenerated synaptic contacts between them will be reconstructed with a computer. Growth of individual neurons will be studied during development and compared with regeneration. An understanding of the mechanisms for accurate regeneration in the leech central nervous system might suggest ways that nerve regeneration in higher animals including humans could be made more reliable and precise.

A basic property of sensory systems is the opponent, center-surround organization of individual receptive fields of neurons. This organization results from the convergence of inputs from separate sensory cells, some of which contribute excitation, and others contribute inhibition. A single sensory cell in the leech synapses with a motoneuron involved in a simple reflex. This sensory cell generates an opponent receptive field in the motor neuron by means of conduction block and an inhibitory interneuron. Experiments being conducted under this award use electrophysiological recording and stimulation, marking of cells with intracellular tracers, laser microsurgery and electron microscopy to characterize the distribution and nature of synaptic contacts involved in the reflex connection and to analyze integration of those inputs by postsynaptic neurons. These experiments should reveal activity-dependent mechanisms that influence qualitative properties of the mechanosensory receptive field and the reflex connections of which it is a part. They will also help understand how postsynaptic cells such as motor neurons integrate sensory signals to reach threshold for firing action potentials and provide insight into the mechanisms by which cell geometry, particularly axonal and dendritic branching, influence neural signalling.

A major problem of neuroscience is to understand mechanisms for behavioral recovery following neural injury. Our collaborative work supported by this grant has provided the first demonstration that capacity for non-associative learning is restored by regeneration of neuronal connections, that a specific connection is crucial for sensitization, although return of function does not immediately follow reconnection, and it suggests new mechanisms for non-associative learning. It is the basis for the proposed cellular and behavioral studies on loss and recovery of the capacity for sensitization following damage to central circuits. Recent work indicates that vertebrates and invertebrates share fundamental mechanisms for axon growth and guidance in addition to neuronal function. In leeches we combine in-depth work on modulation of defensive shortening from one laboratory and work on synapse regeneration by sensory neurons and interneurons including the S-cell from the other. Thus we have shown that the capacity for sensitization of reflexive shortening requires the S-cell, which is restored by regeneration of one of the S-cell's synapses, but with a delay. Proposed experiments will give key information about cellular changes that underlie learning as related to mechanisms for nervous system recovery from injury. Advantages of leeches for such studies include (1) their identifiable neurons, able to re-establish specific connections, (2) selective laser-cutting of single axons in living animals, and (3) stable recordings from identified neurons in behaving animals. Experiments will determine (1) mechanisms for axotomy-induced loss of capacity for sensitization, (2) mechanisms that restore the full capacity for sensitization after injured S-cells reconnect, and (3) the relationship between regulation of functional geometry and repair of intersegmental sensory projections. Methods will include electrophysiology, injection of intracellular markers, laser microbeam axotomy, behavioral testing of leeches including semi-intact preparations, histochemistry, immunocytochemistry, immunoblotting, and confocal and electron microscopy. The studies will reveal basic mechanisms for restoration of plastic properties of the nervous system after injury while at the same time provide insights into the cellular circuitry for non-associative learning.

DESCRIPTION (Verbatim from Applicant's Abstract): A major problem in neurobiology concerns the mechanisms by which damaged neurons may re-grow axons and form selective connections to restore function. We propose to test the role of migrating microglia in successful axonal regeneration that follows injury to the leech nervous system. Migration of microglia occurs promptly, accounts for the increase in cell numbers at the lesion, and appears influenced by nitric oxide synthase activity, which is rapidly up regulated at the lesion. Microglial migration is also affected by applied electric fields of a size consistent with injury currents we measure at the lesion. Evidently microglia deposit laminin, a component of the extracellular matrix that promotes axon growth. The leech is particularly advantageous for these studies because (1) its ganglia contain identifiable neurons capable of regenerating specific connections following axotomy, (2) individual microglia can be tracked and their movements charted minute by minute, and (3) adult and embryonic nervous systems can be manipulated and examined both in vivo and in vitro. Experiments that interfere with and block accumulation of microglia at injury sites will test the role of microglia in sprouting and regeneration. Immunocytochemistry has shown abundant laminin transiently in the embryonic leech nervous system along axon pathways. Following injury to the adult nervous system, laminin reappears (first in patches at the lesion, later in streaks) in advance of axons. Whether the microglia that migrate toward the site of the lesion produce the laminin, as in vitro, will be determined using antibodies and in situ hybridization with our riboprobes for laminin. We will determine whether the streaks of laminin, which may guide regenerating axons, mark the paths of migrating microglial cells; fluorescently labeled microglia will be tracked in living preparations using low-light video microscopy. Alterations of nitric oxide levels and its synthesis and of electric fields such as those generated by the lesion will determine the actions of nitric oxide and fields in regulating microglial migration. These studies will clarify the roles of microglia and of laminin in axonal regeneration following injury, and may suggest strategies for achieving equally successful axonal repair in the mammalian nervous system.

DESCRIPTION (Adapted From The Abstract Provided By Applicant): This is a renewal application for a longstanding training program that is now focussed, providing pre-doctoral and post-doctoral trainees with intensive research experience in the areas of synapses, ion channels and transduction, while ensuring that the trainees have a thorough grounding in the basic neurosciences. The approaches involve the use of advanced molecular biological, physical, chemical, electrical, mathematical, and computational techniques to study fundamental questions in these closely related areas of cellular and molecular neuroscience. Pre-doctoral trainees will enter the program after taking an interdisciplinary group of graduate courses and specialized courses tailored to their needs and after selecting a mentor on the training faculty. Under the mentors direction they will design and execute an original research project, culminating in a Ph.D. degree. Post-doctoral trainees are offered a choice of research topics from which they develop their own independent projects. All trainees receive much individual attention from program faculty and are exposed to a variety of pertinent research activities. Trainees give research presentations and participate in seminars and journal clubs with a large and closely interacting group of neuroscientists. The trainees also take part in career and ethics workshops and gain supervised teaching experience. The aim of this training program is to produce a cadre of highly qualified scientists, capable of working at the forefront of their respective research areas, while being broadly knowledgeable in basic neuroscience and able to communicate effectively. The trainees will be qualified for diverse advanced positions in the biomedical sciences, ranging from academic to those in private industry and government. Accepted trainees must have strong academic credentials, with a clear interest in scientific inquiry. Support is requested for 2 pre-doctoral, 2 post-doctoral, and 4 shortterm (summer medical student) trainees. The neuroscience training program is conducted by 19 faculty members, each actively involved in teaching and research. These faculty have advanced laboratories, and are well funded through individual research grants. Primary research and training facilities are in the Medical Science Building at the Univ. of Miami School of Medicine.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] This is a renewal application for a long-standing, highly focused training program that provides predoctoral and postdoctoral trainees with intensive research experience in the areas of synapses, ion channels, and transduction, while ensuring that the trainees have a thorough grounding in the basic neurosciences. The approaches involve the use of advanced molecular biological, physical, chemical, electrical, mathematical, and computational techniques to study fundamental questions in these focused areas of cellular and molecular neuroscience in which the 20 training faculty have expertise. Predoctoral trainees will enter the program after taking an interdisciplinary group of graduate courses and specialized courses tailored to their needs and interests. Under a mentor's direction they will do original research culminating in a Ph.D. degree. Postdoctoral trainees are offered a choice of research projects from which they develop their own independent research. All trainees receive much individual attention from program and allied faculty. Training involves a broad variety of research activities including presentations and participation in seminars and journal clubs with a large and closely interacting group of neuroscientists. The trainees also take part in career and ethics workshops and gain some teaching experience. The aim of this training program is to produce scientists of high quality, capable of working at the forefront of their research area, broadly knowledgeable in the basic neurosciences, and able to communicate effectively. The trainees will be qualified for diverse advanced positions in the biomedical sciences, ranging from the traditional academic to those in private industry and government. In keeping with the rigorous nature of the program, we seek first- rate applicants. Predoctoral candidates must be strong academically, with demonstrated interest in, and aptitude for, scientific inquiry. Postdoctoral candidates with Ph.D.s must have completed a high quality research project in a relevant discipline; individuals with M.D. degrees are actively recruited. Support is requested for 2 pre- and 2 postdoctoral trainees and short-term training for 4 medical students. The focused training forms the basis for understanding many diseases and disorders of the nervous system, including neurodegeneration, channelopathies, and dysfunction from genetic mutation. By training neuroscientists for research in these critical areas, the program is expected to contribute substantially to public health. [unreadable] [unreadable] [unreadable]