Ronald Kalil - US grants

The research proposed here is designed to clarify certain aspects of the structure and development of the retino-geniculo-cortical pathway in the cat. Four sets of experiments are planned: 1) Possible neurotransmitters in the retinogeniculate pathway will be investigated using light and electron microscopic immunohistochemistry and transmitter-specific retrograde labeling; 2) The effects of impulse blockade on the morphological development of retinogeniculate synapses will be studied in kittens that are reared with intraocular injections of tetrodotoxin; 3) Golgi methods will be used to reveal the effects of monocular deprivation and deafferentation on the dendritic development of neurons in the dorsal lateral geniculate nucleus (LGN); 4) The projection of the LGN to the lateral suprasylvian visual area in newborn and early postnatal kittens will be mapped using horseradish peroxidase (HRP) retrograde tracing methods, and it will be determined in adult cats that have had visual cortex removed at birth whether LGN relay cells that project to the lateral suprasylvian visual area receive direct synaptic input from the retina. The long-term objective of this research is to improve understanding of some of the mechanisms underlying the effects of visual deprivation and amblyopia, and in addition to shed some light on the modes of reorganization employed by the visual system to compensate for damage. These goals are approached by studying normal structure and development in concert with experiments designed to measure the direct effects of abnormal visual experience or brain injury.

The rapid retrograde degeneration of neurons in the lateral geniculate nucleus (LGN) following damage to the visual cortex in adult mammals precludes subsequent axonal regeneration and restoration of function. In young animals of many species, LGN neurons also degenerate after damage to the visual cortex but, in contrast to adults, some neurons are spared and are able to establish new pathways resulting in remarkable behavioral compensation. Recent evidence suggests that basic fibroblast growth factor (bFGF) may prevent neuronal death. However, the lack of a suitable delivery system for administering trophic factors or their genes to non-dividing neurons has been a major obstacle in determining their efficacy in vivo. This Small Grant for Exploratory Research will permit the development of a Herpes simplex viral vector carrying the bFGF gene. This vector will be used to determine whether the production of bFGF in LGN neurons will prevent their degeneration after damage to the visual cortex in adult rat. These studies have the potential for improving the technology for gene therapy not only for treating damaged neurons in the visual system, but also neurons affected by damage, disease or genetic abnormality in any system in the mammalian brain or peripheral nervous system.

Postnatal Neurogenesis in the Mammalian Brain
IBN # 9809905
Ronald Kalil

Lay Abstract

For many years it has been thought that almost all of the neurons in the brain are produced before birth and that few, if any, new neurons are generated thereafter. Although exceptions to this "rule" have been reported in some specialized regions of the brain such as the hippocampus, the generation of new neurons in these germinal regions follows the same set of steps that occur prenatally. This suggests that the germinal zones found in the adult brain may be specialized "holdover regions" from embryonic development. Recently, however, it has it been reported that precursor cells, those cells that produce the neurons and other cell types in the brain during the development, can be isolated from the spinal cord and other areas of the mature brain that are neither near to nor associated with specialized germinal zones. These recent findings suggest that neuronal precursor cells may be distributed widely in the brain, and this raises the possibility that under appropriate conditions these precursor cells may be induced to generate new neurons.

In a previous series of experiments, Dr. Kalil has shown that when neurons in young brains are injured by cutting their axons, which disconnects these neurons from their target cells, that most of the injured neurons die, but that some of them appear to survive. His work also has demonstrated in adult brains that almost all neurons that become disconnected from their target cells quickly die. However, it appears that this cell death can be reduced significantly if specific proteins, known as neurotrophic factors, are administered soon after the injury.

In this project, Dr. Kalil will extend his earlier research by investigating whether neurons in young and adult brains that appear to have survived after having had their axons cut, may instead be new neurons that have been produced in response to the injury. Dr. Kalil will determine if new neurons have been generated in response to injury of the brain by using a marker that will label the DNA only in those cells that have divided after the injury. He will combine the DNA labeling of cells that have divided with staining by specific antibodies that recognize only neurons, in order to identify whether cells that have divided are nerve cells or some other type of brain cell. Using the same methods, Dr. Kalil also will investigate whether the administration of neurotrophic factors to the brain will help to promote the generation of new nerve cells after injury. If the results from this project demonstrate that the brain is capable of generating new neurons in response to injury, they will reveal that the brain retains a greater degree of plasticity after birth than has been thought possible. This will open new opportunities for developing tools that take advantage of this plasticity to assist the brain in repairing itself after being damaged.

DESCRIPTION (provided by applicant): Multi-photon microscopy is perhaps the most important advance in fluorescence microscopy since the introduction of the confocal microscope. Multi-photon microscopy allows visualization deep into structures and permits this visualization to be carded out over extended periods of time with minimal damage to the specimen. Among the advantages provided by multi-photon microscopy are: (a) no out of focus bleaching of fluorophores; (b) deeper optical imaging, because of reduced out of focus fluorophore excitation and decreased scattering of incident wavelengths; and (c) reduced phototoxicity for living specimens. This application requests funds to purchase a: 1.) BioRad Radiance 2100 multi-photon System; 2.) Coherent Mira 900-F tuneable Ti:S laser and Verdi 8 watt ND:YVO4 laser; and 3.) Nikon E-800 upright microscope and a Nikon TE-2000U inverted microscope. The Radiance 2100 multi-photon system has been selected for many reason, among them being a scanhead that can be moved with relative ease from one microscope stand to another, allowing the imaging system to be used with an upright stand for in vivo imaging or with an inverted stand for in vitro imaging. This is an important consideration in this application, because the multi-photon system will be installed in a campus-wide microscopy facility and serve a wide-range of users with different research interests and needs. The Radiance 2100 multi-photon imaging system will be housed in the W.M. Kcck Laboratory for Biological Imaging. The Laboratory was established in 1993 with a gift from the W. M. Keck Foundation, and is equipped with a BioRad MRC 1024 confocal microscope; a single-photon microscope with a mixed gas, krypton/argon laser, capable of three-line excitation. The Keck Laboratory is actively managed, supported by user fees, and is the only confocal microscopy service resource on the UW-Madison campus. The multi-photon imaging system will be operated and maintained by the Keck Laboratory, and access to the imaging system will be open to all qualified users, following well-established procedures that have functioned successfully since the inception of the Laboratory. Currently, the Laboratory provides more than 50 faculty and their students in different areas of biology the opportunity to use confocal microscopy in their research. Among the broad research areas represented by these faculty and students are: agronomy, animal science, biomedical engineering, cell, developmental, and molecular biology, genetics, neuroscience, nutritional sciences, oncology, pathology, reproductive, cardiac, and muscle physiology, etc. The research programs of many of these users will be advanced significantly if a multi-photon imaging system also is available to them in the Keck Laboratory. The acquisition of the BioRad 2100 multi-photon imaging System and associated equipment, as described above, will complement the Kcck Laboratory's BioRad MRC 1024 single-photon confocal microscope, and it will open new avenues of investigation for biological scientists from across the UW-Madison campus.

DESCRIPTION (provided by applicant): In 1993, the W.M. Keck Foundation awarded the first grant to UW-Madison to establish a Laboratory for Biological Imaging (www.keck.bioimaging.wisc.edu). The Laboratory acquired a Bio-Rad MRC 1024 confocal microscope, and began immediately to offer investigators from across the UW campus the opportunity to incorporate confocal imaging in their research. Eleven years later, with an award from NCRR, the Laboratory added a Bio-Rad Radiance 2100 MP confocal/multiphoton microscope, making open source multiphoton microscopy available for the first time at the UW-Madison. In the last 12 months alone, nearly 60 laboratories at UW-Madison, representing approximately 20 broad research areas in the biological sciences, have used the Laboratory's microscopes. Now the Laboratory wishes to replace its original Bio-Rad microscope with a new microscope. While the MRC-1024 has been a capable instrument for 15 years, it now needs repairs almost monthly, and compounding this problem is Carl Zeiss's decision not to offer a service contract for the MRC- 1024 after September of last year. In addition, the MRC-1024 is obsolete by contemporary standards. This is reflected by a reduction of over 60% in usage of the MRC-1024 during the past 12 months, as the Laboratory's users shift their work to the Radiance 2100 MP. Unfortunately, the Radiance cannot accommodate all of those who wish to use it, creating significant delays for some users. In addition, many users operate the Radiance 2100 MP solely for confocal imaging, compromising the microscope's primary role as a multiphoton instrument. In selecting a replacement for the MRC-1024, microscopes from Leica Microsystems (TCS SP5), Nikon (Eclipse C1Plus), Zeiss (LSM 510 META ) and Olympus (FluoView FV1000) were evaluated to determine which instrument would be the best suited for operation in a multi-user imaging laboratory. After comparing each of the systems mentioned, working directly with each, and reviewing their reliability and service records, we have concluded that the Olympus FluoView FV1000 offers the best balance of flexibility, ease of use, and performance for operating in a Laboratory that serves investigators with a diverse range of research projects and experience. The FV1000 requested will be equipped with six laser lines, two standard PMT collection channels, and two spectral detectors. The spectral detectors will enable users to unmix spectrally overlapping signals, a powerful tool for reducing interference by caused by autofluorescence. Separating spectrally overlapped signals also will provide increased flexibility in designing experiments e.g., using eGFP and eYFP in the same experiment, or in imaging simultaneously multiple fluorophores. PUBLIC HEALTH RELEVANCE: This application request funds to purchase a new confocal microscope that will be used by eight biomedical research scientists. Seven of these scientists will use the microscope to study processes that are involved in various diseases such as cancer, anemia, and cardiac aging. The remaining scientist will employ the microscope to study how growing axons in the embryonic brain make proper connections to ensure normal brain development.

This award is funded under the American Recovery and Reinvestment Act of 2009(Public Law 111-5). The project funds a Small Grant for Training and Research in Neuroscience and Public Policy at the University of Wisconsin-Madison (UW-Madison). Three graduate students are supported annually who are engaged in integrated training and research in neuroscience and public policy in the recently established Neuroscience and Public Policy graduate program (N&PP) at UW-Madison. The N&PP is a dual-degree graduate program that leads to a Ph.D. degree in neuroscience, granted by the Neuroscience Training Program (NTP) and a Master of Public Affairs degree (M.P.A.), with an emphasis on Science and Technology Policy, awarded by the La Follette School of Public Affairs. The Program is based on two strongly held beliefs: (1) that sound science and technology policy is essential for the well being of society; and (2) that a step toward ensuring such policy is to train future scientists who are informed about the making of public policy and are prepared to participate in doing so.

The N&PP brings together faculty from the NTP and the La Follette School to train students for a Ph.D. degree in neuroscience and a Masters degree in public affairs (public policy). The training is accomplished in a program that integrates classroom and laboratory research training in neuroscience with a classroom-based education in public policy. In addition to fulfilling all of the requirements that have been established by the NTP and the La Follette School for the Ph.D. degree in neuroscience and the M.P.A. degree, respectively, N&PP students also are required to take the Neuroscience and Public Policy Seminar, which meets monthly, during each of the years that they are enrolled in the N&PP. Thus even after students have completed the requirements for the M.P.A. degree, typically by the end of the third summer, and are working full-time in the last two years of the Program on their doctoral research they maintain an involvement with issues related to public policy. N&PP students also are required to write a critical paper on a topic that bridges neuroscience and public policy as part of the Preliminary Examination for the Ph.D. degree, and complete a summer internship in an agency or organization that is directly involved in science policy.

The N&PP trains future neuroscientists who will have strong research and public policy skills. Currently, there is no other integrated graduate program in the country with goals comparable to those of the N&PP. It is anticipated that one of the major impacts of the N&PP nationwide will be to serve as a model for other institutions who decide to develop similar graduate programs that will integrate training in public policy with neuroscience, genetics or other appropriate biological or physical sciences.