George D. Mower - US grants

The goal of our research program is to understand the causes and underlying mechanisms of amblyopia. Our systemmatic comparisons of animal models of deprivation (monocular, binocular) and non-deprivation (strabismic) amblyopia have revealed significant differences between the two etiologies in terms of physiological and behavioral deficits. It appears that two independent processes are involved in amblyopia, one related to binocular competition and another related to abnormal stimulation per se. These two processes occur to different extents in deprivation and non-deprivation amblyopia and the major deficits in non-deprivation amblyopia appear to result from abnormal stimulation. In the present proposal we will extend these results in several new directions. One will be to more precisely specify the environmental cause of non-deprivation amblyopia. In these studies, we will directly explore the role of preferential eye usage and the role of a defocused image in the strabismic eye in producing physiological deficits in single cells in visual cortex (VC) and the lateral geniculate nucleus (LGN). A second aim will be to assess the role of anatomical loss in the geniculo-cortical pathway (as indicated by transynaptic autoradiography) - in producing physiological deficits in VC and LGN. These studies will use several new animal models which will allow us to more directly define the relation between structural and functional deficits. Our final aim will be to directly assess the relationship between different physiological deficits in VC and LGN and the severity of behavioral amblyopic deficits (as indicated by contrast sensitivity). These studies will significantly improve our understanding not only of the underlying mechanisms of amblyopia but also better define the etiological basis of th disorder with paradigms that are directly applicable to clinical management.

The goal of the proposed research is to define the underlying mechanisms that control the postnatal critical period for cortical development in kittens. The cat visual system is well suited for the analysis of cortical plasticity as recent studies indicate that environmental input not only directs on-going development, but also plays a role in activating the critical period itself. Specifically, rearing in total darkness prolongs the critical period in cats well beyond the normal age limits. We believe that this preparation provides an ideal experimental model, when compared with age matched normals, for isolating the neurological factors that correlate with functional plasticity. We will compare normal and various types of deprived kittens in studies directed at the following specific aims: 1) To define the role of environmental input in the onset and duration of the critical period: Electrophysiological studies will directly determine how the elimination of input, and the delayed introduction of input alter the normal time course of cortical plasticity. 2) To define the relationship between anatomical and electrophysiological plasticity: Trans-synaptic autoradiography and electrophysiological recordings will be used together to specify how the environment effects afferent input to the cortex. 3) To identify underlying factors that correlate with the developmental state of physiological plasticity. One approach will be to examine the effect of environmental input on the development of specific neurotransmitter systems. A combination of receptor binding, immunohistochemistry and biochemistry will be used to provide a broad range analysis of transmitter systems. A second approach will be to examine whether visual input can affect gene expression. The kitten cortex serves as a well studied and easily manipulated neurological model. Our proposed multidisciplinary approach directly addresses the ways in which the environment controls the physiology, anatomy and biochemistry of the developing nervous system.

The aim of the proposal is to identify candidate genes that control neuronal plasticity during postnatal critical periods in neocortex. The proposed studies will utilize the most understood model of neuronal plasticity, the postnatal critical period in visual cortex. To date, studies aimed at identifying molecular mechanisms of visual cortical plasticity have concentrated on the signaling pathways from the cell surface to the nucleus. Dr. Mower's studies will extend this analysis to the output of transcriptional processes in the nucleus, the changes in gene expression which produce the long term structural and functional changes that underlie neuronal plasticity. The strategy capitalizes on the well- established finding that rearing animals in total darkness slows the entire time course of the critical period and prolongs neuronal plasticity far beyond its normal age limits. Prior electrophysiological results in Dr. Mower's laboratory indicate that the effect of dark rearing is to slow the entire time course of the critical period, such that at young ages normal animals are more plastic than dark reared, while at later ages dark reared animals are more plastic. Thus, a stringent criterion is that genes that are important for plasticity in visual cortex will show differences in expression between normal and dark reared animals that are of opposite direction in young vs. older animals. In preliminary work, Dr. Mower's laboratory has completed a differential display PCR (ddPCR) screening of all of the expressed genes in the visual cortex of normal and dark reared animals to directly identify candidate plasticity genes (CPGs) which are differentially expressed according to the above criterion. A manageable number (20) of CPGs were identified. These CPGs will be confirmed by northern blots of visual and frontal cortex from normal and dark reared animals, cloned and sequenced to determine homology to known genes, and analyzed at a cellular level by in-situ hybridization studies. Future uses of the candidate plasticity genes will include experiments to block their expression and determine effects on cortical plasticity in-vivo and in-vitro. The application of differential screening techniques is a novel experimental approach to analyzing critical period plasticity. The identification of effector genes that control development and plasticity would be a major leap in the state of knowledge regarding molecular mechanisms of the visual cortical critical period and neuronal plasticity. Such information is essential to our understanding of processes such as brain development, learning, and memory.

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to identify candidate genes controlling neuroplasticity (CPGs) in visual cortex. Dark rearing slows the entire time course of the visual cortical critical period and therefore provides a means to dissociate changes associated with the state of plasticity from changes associated with maturation. A' differential display PCR screening, by this laboratory, of all of the expressed genes in the visual cortex of normal and dark reared cats at 5 and 20 weeks identified 23 CPGs. CPGs fall into 2 classes: "plasticity"' genes whose expression is enhanced in age/rearing conditions where neuroplasticity is maximal, and "anti- plasticity" genes whose expression is opposite. One novel CPG that was identified is Mund 3-3 ("anti- plasticity"), which plays an essential role in vesicle release at glutamatergic synapses. A novel "plasticity" CPG is disabled-1 (Dab-1), a gene that plays a central role in the migration of cortical neurons. The project will address the following specific aims: 1. To test the hypothesis that CPG gene mutation will alter the time course of the physiologically defined critical period in mouse visual cortex. Electrophysiological and immediate early gene expression methodologies will determine the effect of Mund 3-3 and Dab-1 mutation on susceptibility to monocular deprivation. 2. To test the hypothesis that "plasticity" and "anti-plasticity" CPGs will show distinct temporal associations with known laminar specific developmental events during the critical period in visual cortex. In situ hybridization and immunohistochemistry will be used to compare "plasticity" and "anti-plasticity" gene/protein expression in layer IV and extragranular visual cortical layers as a function of age and dark rearing. 3. To verify the identity of previously unsuspected CPGs indicated by the gene screen. Our screen has implicated novel CPGs with documented neurological functions. In addition to Mund 3-3 and Dab-1, Plasticity Related Gene 1 (PRG-1) which influences axonal outgrowth, and two genes associated with Ras mediated cytoskeletal reorganization (Chimaerinl and Abl interacting protein 2) are tentatively identified. The identification of effector genes which control the visual cortical critical period would be a major step in unraveling molecular mechanisms of neuronal plasticity. Such information is essential to understanding processes such as neuronal development, regeneration, learning and memory and would further open the possibility of manipulation of nervous system neuroplasticity for therapeutic benefit. [unreadable] [unreadable] [unreadable]