Gerald Fischbach - US grants

Seven investigators with career-long interest in the developmental biology of the synapse propose to extend their individual efforts by collaborations focused on the formation of synaptic connections and the degree to which synaptic connections change over time in adult animals. The work has five broad aims: (1) elucidating mechanisms that initiate axon growth and then terminate axon growth after synapses form; (2) identifying structures and molecules that influence the specificity of synaptic connections; (3) characterizing signals that promote the differentiation of synapses; (4) characterizing the molecular components of the postsynaptic membrane and the manner in which they are regulated; and (5) directly observing changes in identified synapses in adult animals over periods of weeks to months. We have chosen to carry out this work in vertebrates - generally mammals - because of our concern that the findings be applicable to the human nervous system and to human neurological disorders. The significance of this work lies in its potential contribution to the solution of one of the major problems confronting modern neurobiology: how the formation and malleability of synapses explain the remarkable adaptive abilities of the nervous system. Knowledge gained about normal development and plasticity of synapses will also advance our understanding of pathological situations in which synaptic connections fail to form properly or degenerate prematurely.

Our long-term goal is to define the electrophysiological, morphological and biochemical correlates of synapes formation. Most experiments are performed with cultures of cells dissociated from embryonic chick spinal cords ciliary ganglia, sensory ganglia and muscle. In nerve-muscle co-cultures, patch-clamp microelectrodes will be used to describe parameters of acetylcholine (ACh) release from growth cones and newly formed synapes, and also to monitor the accumulation of ACh receptors and acetylcholinesterase (AChE) in the postsynaptic complex. Reverse-phase HPLC and other chromatographic techniques will be used to purify factors from neural tissue that induce the synthesis of ACh receptors and AChE. Antibodies raised against purified material will be used to localize the activity and to study its role in normal development. the influence of competing neurons and of impulse activity on the stability of synapses will be studied in a new microculture system. In spinal cord-sensory neuron co-cultures, the specificity and function of synapses formed on identified motoneurons (labeled in vivo with a fluorescent probe via retrograde axonal transport) will be tested. Patch-clamp techniques will be used to assay transmitter condidates, to map the distribution of chemoreceptors, and to analyze the inonic basis of presynaptic and postsynaptic inhibition. Information about the function, stability and specificity of newly formed synapses has import for developmental neurological defects, for neural mechanisms that underlie learning and memory, and for mechanisms of degeneration and regeneration in the mature brain.

This is a response to a NIDA RFA for the establishment of a Neuroscience Network. This proposal contains a series of highly interdigitated projects, connected both scientifically and technically. The major theme running through all four projects is the action of neurotrophic factors and cytokines in neural systems relevant to drug abuse. A second major theme running through three of the four projects is modulation of mesolimbic dopamine systems by neurotransmitters and trophic factors. A third theme binding two of the projects closely together is the study of cholinergic-dopaminergic interactions. The Network involves entirely new project from two labs already involved in drug abuse research and recruits the laboratories of two well established neuroscientists to drug abuse research for the first time. A major connection of all the investigators is their commitment to the use of molecular and cellular tools in the service of understanding brain and behavior. The investigators, who comprise a highly collegial group, well known to each other, are particularly enthusiastic about the communications core which will facilitate their work together. This RFA has provided an opportunity for the group to develop synergistic interactions across physical distance that will speed their investigations of fundamental processes in the nervous system relevant to the actions of drugs of abuse.

The main goal of this project is to study the regulation of nicotinic acetylcholine receptors (nAChRs) in the CNS by neuregulins, a new family of neurotrophic factors that are present in cholinergic neurons throughout central and peripheral nervous system. We will also determine if neuregulin induced change in the number of distribution of functional nAChRs alters synaptic transmission in the circuits described. Neuregulins will be studied in vitro by adding them to neuronal cell cultures, and they will be studied in vivo in mutant mice that exhibit diminished neuregulin expression. To maximize the generality of our results, we will study neurons dissociated from the Medial Habenula (MHb), the Interpeduncular Nucleus (IPN), the Substantia Nigra, (SN), and the Ventral Tegmental Area (VTA). Each region offers advantages for studies of nAChR regulation. In each case, the nuclei can be dissected easily from embryos and neonatal animals, the cells can be maintained in spars cell culture, a subpopulation of the neurons receive cholinergic synaptic inputs, and the neurons express a diverse array of nAChRs. In vitro cell culture experiments are described in the three Specific Aims. In vivo studies that utilize fresh slices prepared from normal and mutant mice are described in Collaborations. We emphasize microelectrode recordings of ACh induced currents because we want to study regulation of functional receptors. However, ligand binding and receptor subunit gene expression will be used to confirm and extend results in some cases. In each series of experiments, we will begin with the relatively well studied MHb-IPN system, but results will be extended to Septal-MHb, and PPT-SN/VTA interactions.

We are requesting funds for major alterations and renovations of existing research space at the College of Physicians & Surgeons of Columbia University for the purposes of conducting basic and translational research in the field of vascular diseases. The College has recently established a Center for Vascular Biology in recognition of the importance of vascular diseases to our patient population, to exploit recent advances in the understanding of mechanisms underlying vascular disease, and to recognize the enormous opportunities for basic and translational research that have resulted. The Dean has identified a highly integrated team of first-rate investigators to serve as core faculty for the vascular biology effort, and seeks to establish a physical environment of 5,000 square feet of laboratory space supportive of the special programmatic requirements of this new research initiative in our Medical Center. The core group of four investigators represents the Departments of Surgery, Pathology, Center. The core group of four investigators represents the Departments of Surgery, Pathology, Medicine, and Physiology & Cellular Biophysics, and forms the nucleus for a large collaborative group drawn from the Departments of Anesthesiology, Neurological Surgery, Pediatrics, Pharmacology, Radiology, and the School of Dental and Oral Surgery. The Center will target four key thematic areas in vascular biology including diabetic vascular dysfunction, cerebrovascular disease, reperfusion injury and the biology of thrombosis. This program already accounts for approximately $1.3 million in annual direct costs from NIH (further direct costs from the Juvenile Diabetes Foundation and Heart Association are approximately $571,350/year), in addition to approximately $1.02 million/year in pending Federal grants. The larger collaborative group of investigators accounts for approximately $4.8 million in annual direct costs on Federal grants. Exciting observations have emanated from the core group of investigators, including new insights into mechanisms underlying diabetic vascular disease, cerebral ischemic and amyloid angiopathy, and the biology of thrombosis. Core members of the Center for Vascular Biology are now working in non- contiguous places within the University, using space designed for research activities quite distinct from those required for their collaborative research program in Vascular Biology. The proposed changes in the research environment will facilitate our ability to upgrade, consolidate and modernize the physical plant into efficiently designed and operational laboratories with specialized shared core facilities and laboratory modules. This will result in more effective use of our laboratory space, and will allow us to efficiently perform studies which can only be done with considerable difficulty at the present time. These facilities will be used exclusively for the purposes related to the research as outlined by the specific aim of our federal and non-federal grant proposals, and the research training (teaching) of pre-doctoral and postdoctoral fellows for at least the next 20 years.

The machine shop in the Department of Neurobiology currently consists of two lathes, a milling machine, a bandsaw and two drill presses. It is managed by Mike Lafratta. This core will bring together Mike's forty years of experience he has in building and designing neurobiological equipment, offering more specified techniques that could ever be supplied by a machine shop serving the school's departments. His experience is not limited to building metallic or plastic machinery, for he has taken advantage of years of development of equipment by a large number of exceptional scientists. Many of the devices he makes have gone through many model adjustments over the years to suit the department's needs. It is rare when he requires anything but a rough sketch to design what is needed for a lab. Due to his expertise, he is able to make useful suggestions as to improvements of a particular design. Additionally, he offers knowledge of solving and fixing and mechanical problem encountered in the labs. During the past years, he has provided invaluable service to the numbers of the program project grant by building and adapting countless custom optical components, microscopes for use with brain slices, adaptors for manipulators, perfusion setups, temperature control apparatus, recording chambers and countless other contributions to the research programs of Drs. Bean Clapham, Fishbach and Regehr. The machine shop will be one of the most extensively used by the Core members as they begin new projects related to this program project grant.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We are requesting funds for construction of research space to house the Institute for Cancer Genetics (ICG), a critical component of Columbia University devoted to basic and translational research on human cancer. The space would occupy the 8th and 9th floors of the new Irving Cancer Research Center. The core and shell construction of this building is underway and will be completed by mid 2003. The ICG was established in 1999 as a University Institute devoted to research and teaching on human cancer. The ICG's mission is to elucidate the pathogenesis of cancer and identify targets for diagnosis and treatment of malignant disease, and as such to serve as the main research arm of the Herbert Irving Comprehensive Cancer Center. Dr. Dalla-Favera was named ICG Director and assigned the task of forming the Institute faculty. In less than four years the ICG has grown to include 9 primary research laboratories led by faculty members with appointments in 5 different clinical or basic science Departments. The research activities of the ICG are organized into four programmatic areas: lymphoid neoplasia, breast cancer, brain tumors, and mechanisms of genomic stability. These Programs were chosen to optimize the impact of ICG research in the battle against cancer and to exploit existing areas of strength in other fields of biomedicine at Columbia. As such, each of the four ICG Programs seeks to develop a fertile research environment and the critical mass necessary to achieve significant scientific advances. By any measure, the ICG has been immensely successful in both its research and teaching activities. This is manifested in its increasing funding base from peer-reviewed grants, which has grown from ~$1.5 million in June 1999 to ~$5.4 million of currently approved funds (annual direct costs), including nine Ro1 grants, a P01 Program Project grant, and a Cancer Biology Training grant. When the ICG was established in 1999, it was assigned to ~15,500 square feet of swing space in the Russ Berrie Science Pavilion. This was intended to be a temporary arrangement until the ICG was moved to its permanent home in the future Irving Cancer Research Center. The proposed relocation of the ICG to this site will provide multiple b for existing ICG laboratories that have grown in personnel and funding; ii) the recruitment of two new faculty necessary for completion of the ICG research program; and iii) laboratory space to host state-of-the-art core facilities available to all Columbia University investigators (i.e., Genomics, Proteomics, Cytogenetics, and Pathology). Finally, relocation of the ICG to the proposed facility represents a crucial first step in the consolidation of cancer research activities at Columbia in a single dedicated building, the Irving Institute of Cancer Research. These facilities will be used exclusively for purposes related to the research outlined in the specific aims of our federal and non-federal grant proposals and for the research training/teaching of predoctoral and postdoctoral fellows for at least the next 20 Years. [unreadable] [unreadable]