John R. Kirn - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Unlike most mammals, birds retain the capacity to generate new neurons in adulthood. In the canary, a telencephalic song control area (HVC) incorporates new neurons in register with adult vocal plasticity. These cells migrate from their birthplace in the walls of the lateral ventricles into HVC, where they replace other cells that presumably have died. Some new neurons become interneurons, while others extend axons roughly 3 mm to become projection neurons in the motor pathway controlling learned song. Studies of adult HVC neurogenesis and neuronal death could improve our understanding of vocal learning , adult neural plasticity, and recovery o function following brain damage. This proposal describes experiments which explore; 1) the timetable of the differentiation of connectivity by adult- formed neurons, 2) possible mechanisms controlling the expression of specific phenotypes by these cells, 2) the types of contacts made by new neurons with others in HVC,3) the time course and amount of naturally occurring HVC neuron death, and 4) the potential relationship between hormones, cell division and cell death. The phenotypes expressed by neurons born in adulthood may be rigidly specified at the time of cell division or could be determined later through interactions between neuroblasts and other cells. Early events in the differentiation of cell type among adult-formed neurons will be characterized by treating birds with 3H-thymidine, a cell birth marker, and then retrogradely labelling neurons with multiple axonal tracers injected into HVC targets at various times after 3H-thymidine treatment. This procedure will reveal the time course for cell differentiation and test for the possibility that adult-formed HVC cell types are determined through a process of axon elimination. Moreover, direct soma-soma contacts made by adult-formed neurons with other cells intrinsic to HVC will be identified and quantified by combining the above methods with serial cell reconstructions from 1-2 mu sections. These methods will permit an assessment of whether there is a relationship between the phenotypes of new neurons and other cells in close contact. The timing and magnitude of naturally occurring cell death will be established by labelling HVC neurons with a vital dye and then quantifying cell loss at various survival times. Endogenous hormone levels will be manipulated in some of these birds to assay whether testosterone controls neuron survival. These birds will also be treated with 3H-thymidine prior to sacrifice to determine the impact of hormone levels on cell production in the ventricular zone. An exploration of the mechanisms which determine cell type and the factors regulating cell division/death could provide basic information of relevance to our understanding of the functions and control of adult neurogenesis.

9415123 Kirn Nottenbohm Macklis Many brain disorders, result from the death of brain cells. We do not know how to replace these cells, or even if this is possible. However, death and replacement of brain cells (neurons) occurs spontaneously in the brain of adult birds. The new neurons are born in the walls of the lateral ventricle of the brain. Some of these cells are part of the vocal control system, and are known to be involved in controlling the learning and remembering of song. Since this constant replacement is not preceded or accompanied by overt pathology, it can be thought of as a form of brain rejuvenation. In collaborative research proposed here by Drs. Nottenbohm, Macklis and Kirn, a new technique for inducing selective cell death in the brain will be used and resulting effects on the generation and function of new brain cells will be tested. The questions that will be answered are: 1) Is cell replacement restricted to a few kinds of cells, or will any type of brain cell that dies be replaced by another one of the same kind? 2) Do signals emitted by dying cells play a role in the birth, migration, and survival of the new cells? 3) Does cell replacement affect memory and the acquisition of new memories? The answers to these questions will help reveal the cellular dynamics underlying memory formation, and may help in the development of new approaches to repairing brains and controlling the decrement of function that accompanies aging.***

Unlike most mammals, birds retain the capacity to generate new neurons in adulthood. In the canary, a telencephalic song control area (HVC) incorporates new neurons in register with adult vocal plasticity. These cells migrate from their birthplace in the walls of the lateral ventricles into HVC, where they replace other cells that presumably have died. Some new neurons become interneurons, while others extend axons roughly 3 mm to become projection neurons in the motor pathway controlling learned song. Studies of adult HVC neurogenesis and neuronal death could improve our understanding of vocal learning , adult neural plasticity, and recovery o function following brain damage. This proposal describes experiments which explore; 1) the timetable of the differentiation of connectivity by adult- formed neurons, 2) possible mechanisms controlling the expression of specific phenotypes by these cells, 2) the types of contacts made by new neurons with others in HVC,3) the time course and amount of naturally occurring HVC neuron death, and 4) the potential relationship between hormones, cell division and cell death. The phenotypes expressed by neurons born in adulthood may be rigidly specified at the time of cell division or could be determined later through interactions between neuroblasts and other cells. Early events in the differentiation of cell type among adult-formed neurons will be characterized by treating birds with 3H-thymidine, a cell birth marker, and then retrogradely labelling neurons with multiple axonal tracers injected into HVC targets at various times after 3H-thymidine treatment. This procedure will reveal the time course for cell differentiation and test for the possibility that adult-formed HVC cell types are determined through a process of axon elimination. Moreover, direct soma-soma contacts made by adult-formed neurons with other cells intrinsic to HVC will be identified and quantified by combining the above methods with serial cell reconstructions from 1-2 mu sections. These methods will permit an assessment of whether there is a relationship between the phenotypes of new neurons and other cells in close contact. The timing and magnitude of naturally occurring cell death will be established by labelling HVC neurons with a vital dye and then quantifying cell loss at various survival times. Endogenous hormone levels will be manipulated in some of these birds to assay whether testosterone controls neuron survival. These birds will also be treated with 3H-thymidine prior to sacrifice to determine the impact of hormone levels on cell production in the ventricular zone. An exploration of the mechanisms which determine cell type and the factors regulating cell division/death could provide basic information of relevance to our understanding of the functions and control of adult neurogenesis.

Abstract Naegele NSF 0070352


A Confocal Microscope for Research and Teaching in Biology and Neuroscience

A Zeiss 510 Confocal Microscope will be used for research and training in developmental biology and neuroscience at Wesleyan University. This new confocal microscope will enable the three primary faculty users and members of their research laboratories to study the dynamic movements of cells and proteins in living embryos and to identify intercellular junctions between cells in thick sections of brain tissue. In addition, training on the confocal microscope will be an important component of a Biology department graduate-level course in advanced microscopy, including confocal, immunofluorescence, and electron microscopy. The confocal microscope facility is part of an Advanced Instrumentation Facility in the Science Center at Wesleyan University. This facility already contains scanning and transmission electron microscopes, an adjacent wet laboratory, a room for tissue sectioning, and a computer room with associated digital scanners and other image processing equipment.

The projects to be carried out by major users of this confocal microscope, Drs. Devoto, Kirn, and Naegele, and members of their laboratories, each focus on vertebrate development. The Devoto laboratory will focus on the genetic and molecular guidance cues used by migrating muscle cells in living zebrafish embryos. The Kirn laboratory will address the mechanisms of neuronal replacement and neurogenesis in the brains of adult birds. The Naegele laboratory will study cellular and molecular signals regulating programmed cell death and engulfment of dying neurons in the rodent cerebral cortex and visual system. Occasional use of the confocal is also planned by five additional faculty in the Biology and Molecular Biology and Biochemistry Departments who study a variety of biological problems ranging from the role of transcription factors in embryo development to the cell cycle regulation in yeast. Additional minor use by one extramural group Pfizer, Inc. is planned for 2 days/month. Funds from this extramural group will be a significant source of revenue for long-term maintenance of the confocal, including service contracts.

We anticipate that this confocal microscope and the associated research programs will have a significant impact on research and training at Wesleyan University in the fields of developmental biology and neurobiology. The objective of our science training programs at Wesleyan University is to provide high-quality research experiences for undergraduate and graduate students, as well as postdoctoral fellows and visiting scientists. This hands-on training is of fundamental importance for careers in scientific research, technology, and education. Special initiatives at Wesleyan University advance women and minorities in science, as well as providing access for students with disabilities. It is anticipated that the new confocal microscope will contribute significantly to a basic understanding of how cells in the developing embryo migrate, form connections, and how some undergo programmed cell death as part of their normal developmental plan. These studies will ultimately lead to a better understanding of how the vertebrate brain and body are constructed during development and maintained throughout the life of the organism.

DESCRIPTION: (adapted from applicant's abstract) Neurogenesis persists into adulthood in many vertebrates including humans. An understanding of the factors that control neuron addition and survival may ultimately lead to insights on the functions of adult neurogenesis and suggest mechanisms for brain repair. In adult warm-blooded vertebrates, the most widespread production of neurons occurs in the avian telencephalon. In songbirds, many new neurons are inserted into the High Vocal Center (HVC), a region necessary for the perception and production of learned vocalizations. New HVC neurons replace older neurons that have died. Recent work shows that auditory input is necessary for normal rates of neuronal replacement. Available data suggest that deafening decreases the numbers of HVC neurons incorporated and prolongs their subsequent survival. Proposed work will directly test whether the life span of neurons that have been successfully incorporated into HVC prior to deafening is augmented. Additional studies will determine whether deafening influences the production or early survival of adult-formed neurons. A lesion study will test whether the lateral magnocellular nucleus of the anterior neostriatum (lMAN), an area necessary for song learning, participates in the auditory control of HVC neuron turnover. Playbacks of conspecific song produce increased rates of HVC neuron activity and maximal rates are obtained with playbacks of the bird's own song. Thus, the effects of deafening on HVC neuronal turnover may be due to depriving birds of song-related auditory stimulation. Proposed experiments will systematically deprive hearing-intact birds of access to self-generated auditory feedback and/or conspecific song to test this hypothesis. We will contrast these effects with those following manipulations that alter, without completely blocking, self-generated auditory feedback from singing. Collectively, these studies will explore the sensory requirements for the control of adult neuron addition and loss within a discrete, well-characterized neural system controlling a learned vocal behavior.

DESCRIPTION (provided by applicant): Neurogenesis persists into adulthood in many vertebrates including humans. An understanding of the factors that control adult neuron addition, differentiation and survival may ultimately suggest mechanisms for brain repair. In adult warm-blooded vertebrates, the most widespread neuron production occurs in the avian telencephalon. In songbirds, many new projection neurons are inserted into the High Vocal Center (HVC) to become part of the pre-motor, efferent pathway necessary for the production of learned vocalizations. Thus, studies of the avian brain may address basic questions about the mechanisms permitting widespread neuron addition and, at the same time provide an exceptional opportunity to relate neuron addition in discrete brain regions to a well-characterized behavior-song. Deafening leads to song deterioration and the impact on song decreases with increasing bird age or vocal practice. Correlations between singing history, song stereotypy, motor program stability (defined by reliance of song structure on auditory feedback) and neuronal incorporation established during the prior period of support will be directly tested by manipulation of singing prior to deafening. To further explore the functional significance of adult neuronal replacement, selective manipulation of HVC stem cells will be required. Using retroviral methods, the relationship between birthplace and final destination for HVC cells and neurons destined elsewhere in the telencephalon will be mapped. This work will identify sites of HVC stem cells for future manipulation and provide a fate map of the topographical relationship between proliferative zones and the telencephalon more generally. The first comprehensive analysis of adult-formed vocal control neuron structure and electrophysiological properties will also be conducted using retroviral vectors. Morphological comparisons among adult-formed HVC neurons and new neurons dispersed throughout the telencephalon will provide a first appraisal of the adult avian brain's natural potential to produce and integrate multiple neuron classes. Functional properties of adult-formed HVC pre-motor neurons (visualized by retroviral labeling with green fluorescent protein) and sources of synaptic input will be identified by electrophysiological stimulation-recording work in tissue slices as a step toward understanding how these cells are integrated into the brain. Collectively, this work will test the relationship between cell replacement and song, provide basic information about the avian brain's natural potential for producing diverse cell types, and set the stage for future work aimed at testing the functional consequences of suppressing replacement on learned vocal behavior.

An award is made to Wesleyan University to support the acquisition of a confocal microscope for use by faculty and students across multiple departments and disciplines. The new instrument will profoundly enhance the training environment and be used during many highly interactive laboratory courses taught by Wesleyan faculty to promote STEM career advancement. The proposed microscope will be housed in an advanced imaging facility that includes scanning and transmission electron microscopes. To support dissemination of science to the general public, as exemplified by the large number of articles authored by Wesleyan faculty that are printed in the popular media, data generated by the new system will be used in outreach presentations, science camps, local high-schools, an annual scientific imaging student prize, and similar forums.

Wesleyan boasts an unusually broad portfolio of research topics that will benefit from the new system, including evolution and development of morphology, optogenetic-based control of behavior, cell biology, physiology, developmental neuroscience, and chromosomal dynamics. Each of these areas is experiencing an expansion of research into spatial relationships, driven by advances in microscopy, and by the realization that researchers must study three-dimensional shapes and how those change over time to understand the structure and function of biological systems. Several of the research projects will only be possible because the local availability of the system. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.