Leo M. Chalupa - US grants

DESCRIPTION (adapted from applicant's abstract): The overall objective of the proposed research program is to further our understanding of the functional and structural development of retinal ganglion cells, the neurons that convey all information from the eyes to the visual centers of the brain. Five specific aims are proposed designed to fill major gaps in our current knowledge. As the first specific aim, patch-clamp recordings will be made from developing ganglion cells of the ferret in conjunction with their intracellular filling to allow for the morphological assessment of cells into major classes (alpha, beta and gamma) as well as On, Off and On-Off subtypes. Spontaneous activity patterns will be recorded at three well-defined stages of development and quantitative methods will be used to assess discharge patterns. The aim of this study is to determine whether different classes of cells manifest class-specific spontaneous activity patterns during the developmental period when activity mediated refinements are known to occur at retinorecipient structures. The second specific aim is to determine whether two intrinsic membrane properties studied during the past grant period (speed of recovery from Na-channel inactivation and Ca-mediated potassium currents) relate to the spontaneous activity patterns manifested by developing ganglion cells. The third specific aim is to determine how the visual responses of developing ganglion cells to flashing spots of light correlate with the stratification of dendrites in these neurons. In particular, we want to learn whether immature ganglion cells with dendrites ramifying within both the On and Off sublaminae of the developing IPL respond to the onset as well as the offset of flashing spots of light. The third specific aim is to determine whether the functional circuitry underlying On and Off responses in the developing retina is equivalent to that found in the mature retina. For this purpose, we will assess the effects of APB application on the light-evoked responses of morphologically identified ganglion cells. This glutamate agonist selectively blocks On responses in the mature retina, but preliminary results indicate that both On and Of f pathways can be effectively blocked by this drug in multistratified ganglion cells of the developing retina. The fifth specific aim is to determine whether cholinergic amacrine cells play a role in the formation of segregated On- and Off-cone bipolar cell axonal projections and in the stratification of retinal ganglion cell dendrites. To address this issue we have devised a novel method for eliminating cholinergic amacrine cells from the developing retina. The successful completion of this research program will provide new insights into the development of the mammalian retina, and it will also have implications for elucidating the cellular mechanisms underlying the formation of neuronal connections at higher levels of the nervous system.

The overall objective is to hold a high level tutorial ("school") that will bring together for a 10 day period the leading international experts in the field of visual developmental neurobiology with young faculty, postdoctorals, and graduate students who are interested in acquiring knowledge and expertise in this field. The purpose of the school is two-fold. To provide a relatively informal forum by which the students can interact with established researchers, and to provide an opportunity for exchange of information among the representatives of the leading laboratories.

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.

9723151 CHALUPA In the vertebrate retina the ON and OFF pathways, which signal increments and decrements of light, are anatomically segregated from each other into separate layers at maturity, but not early in development. During the previous period of NSF funding for this project, Dr. Chalupa's laboratory discovered that treating the developing retina with 2-amino-4-phosphonobutyrate (APB), the selective blocker of bipolar cell activity, prevents the normal stratification of retinal ganglion cell (RGC) dendrites into ON and OFF sublaminae in the inner plexiform layer, where synaptic contacts are made between the bipolar cells and RGCs. This finding suggested the hypothesis that activity in the bipolar cells regulates the retraction of RGC dendrites, which are initially present in both strata, or "multistratified." The current project will extend these findings by addressing three specific aims. The first aim is to establish whether the multistratified RGCs, resulting from APB treatment, respond to both the onset and offset of visual stimuli. This would provide functional evidence that blockade of the afferent activity during development results in an intermingling of ON and OFF retinal pathways. The second aim is to determine the temporal relationship between the ingrowth and segregation of bipolar cell terminals and the time course of RGC dendritic stratification. Segregation of ON and OFF bipolar cell afferents prior to the stratification of RGC dendrites would lend further support to the hypothesis that retinal afferents regulate the normal stratification of RGC dendrites. The third specific aim is to investigate synaptic connectivity between developing bipolar cells and multistratified RGCs by using the whole-cell patch-clamp technique to record synaptic activity in these neurons while stimulating their retinal afferents. These recordings will provide a direct test of the hypothesis that such retinal synapses are functional early in development. The successful completion of this research project will further our understanding of the mechanisms underlying the formation of ON and OFF retinal pathways.

animal tissue;

DESCRIPTION (provided by applicant): Continued CORE grant support for the research efforts of vision scientists at the University of California, Davis is requested. In the past five years there has been a major increase in the number of faculty hired with primary research interests in the visual sciences, and a near doubling in NEI funded grants. The CORE has played a major role in this virtual "boom" in the visual sciences at UC Davis. It has enhanced the ability of our faculty to interact in efficient and productive manners, and has called attention of campus administrators to the excellent cadre of faculty devoted to vision research. The present proposal seeks funding for four modules designed to support the needs of our current faculty and to attract new faculty slated for the proposed Center for Vision Research. The modules are: (i) Multi-neuronal Recordings Module directed by Dr. Ken Britten, supports programming and computation needs of our neurophysiologists; (ii) Retinal Cell Culture Facility Module directed by Dr. Martin Wilson, offers tissue culture facilities as well as imaging of retinal tissue; (iii) Microscopic Anatomy Module directed by Dr. Paul FitzGerald provides microscopy and histological services; (iv) Machine Shop Module, headed by Dr. John Werner, offers design, fabrication, maintenance, modification and repair of essential items of equipment not available commercially. Three of the modules funded previously, remain essentially unchanged from the last grant period, since they have proven to be effective and useful resources for the CORE grant faculty. The Machine Shop Module has been added after consultation with the vision researchers on this campus. It was deemed as having the most widespread utility across all of our research areas. The support of the campus administrators for the CORE has been, and continues to be, outstanding. This is reflected by the recent hiring of 10 additional vision faculty. Also, a proposal for a new Center for Vision Research has been approved by the Dean of the Medical School as well as the Dean of Biological Sciences, and is currently under consideration by the Provost, and the Dean of the Medical School has recently pledged $1,000,000 as part of his commitment to vision research on this campus. The research programs funded by the CORE are relevant to furthering our understanding of the basic mechanisms of vision, and they are consistent with our long-term goal of developing effective treatments of myriad visual disorders.

DESCRIPTION (adapted from the Abstract): The goal is to enhance our understanding of the role the hippocampus plays in memory using a naturalistic model, memory for cache sites in food-storing birds, in which certain memory features have become modified during evolution in response to the selective advantage of retrieving stored food caches. This model system is unique in that specific experiences related to memorization have a dramatic impact on volume and neuron number of a distinct region of the brain, the avian hippocampal formation (HF), at a relatively late stage in development, after the young have left the nest. This result raises several questions about the types and amounts of experience needed to trigger these morphological changes and the time course over which they occur. Previous work has focused exclusively on HF in juveniles. However, HF has major connections with other telencephalic regions including archistriatum (ARCHI) and lobus parolfactorius (LPO). Therefore, the determination of whether or not neuronal changes can be detected in HF alone or additionally in other regions, and of what role these regions might play in memory is now important. Also, whether brain changes are restricted to juveniles or whether experience-dependent growth and atrophy occurs in the adult brain must now be tested. Further, because the intensity of food-storing fluctuates seasonally, changes in photoperiod may produce similar effects on brain plasticity. The intent of this research plan is to compare and contrast developmental and seasonal changes in food-storing behavior, memory, and the brain. Experiments will be conducted on food-storing mountain chickadees and black-capped chickadees. Bush tits will serve as a non-storing species control. The aims are: (1) to analyze the effects of food-storing experience and season on brain morphology in adults and juveniles by manipulating photoperiod and the opportunity to store; (2) to examine the nature of experiences needed to trigger growth and attrition by manipulating the amount of seeds stored and duration over which caches are stored; (3) to determine the time course over which changes occur by sampling juveniles with and without food-storing experience at different ages; (4) to assess the relative importance of programmed cell death and cell birth on brain morphology; and (5) to lesion HF, ARCHI,and LPO to assess the role each region plays in memory for cache sites, whether they are the same regions that show morphological change, and whether the effects of lesions are permanent.

DESCRIPTION (provided by applicant): The objective of the proposed research program is to further our understanding of the cellular and molecular mechanisms underlying the formation of specific retinogeniculate projections in the primate visual system. We will focus on the development of the two major functional streams, magnocellular (M) and parvocellular (P), which at maturity are a hallmark of the primate visual system. In Specific Aim 1, we will utilize modern neuroanatomical tracing methods in combination with pre- and postsynaptic molecular markers to learn how the formation of M and P pathways relates to synapse formation in the dLGN. Our working hypotheses are: (i) that eye-specific inputs form in the P pathway earlier than in the M pathway, and (ii) that selective ingrowth of retinal fibers into the P and/or M subdivisions of the dLGN occurs prior to synaptogenesis. In Specific Aim 2 we will define the spatiotemporal properties of retinal activity during the time that retinal axons selectively innervate the M and P segments of the dLGN. If correlated retinal activity is observed at this early stage of development, we will assess the effects of perturbing and also blocking such activity on the formation of M and P retinogeniculate projections using a novel immunotoxin developed in our laboratory. The foregoing experiments will tell us whether or not neuronal activity plays a role in the development of these two functionally distinct pathways. In Specific Aim 3 we will utilize DNA microarrays and in situ hybridization methods to find molecular cues expressed in the developing dLGN during the period when M and P pathways are being formed. The proposed research program will break new ground by increasing our understanding of the mechanisms underlying the formation of M and P pathways which are a distinguishing feature of the primate visual system.