The objective of this project is to study the signal transduction pathways involved in adult cortical plasticity. Our experimental approach will be to genetically inhibit neurotrophic gene expression in the living macaque monkey brain to determine if these trophic factors are essential for cortical remodeling in the visual cortex after depravation of sensory input. Although certain functional properties and circuits become fixed after a critical period early in life, the visual cortex retains the capacity for experience dependent change throughout life. A useful model to explore the mechanisms of this change is remapping of cortical topography following retinal lesions. After retinal damage, cortical receptive fields in V1 shift away from representing the lesioned retina area and cortical representation expands in areas immediately surrounding the damaged retinal region. Changes in receptive fields after focal binocular retinal lesions in the adult Macaque monkey were correlated with cortical remodeling of horizontally projecting axons in V1 months to years after injury. Yet, the signaling mechanisms responsible cortical axon sprouting, and ultimately alterations in cortical receptive fields are unknown. Previous studies demonstrate neurotrophic factors brain derived neurotrophic factor (BDNF) and its receptor TrkB are enriched at the site of cortical scotoma after retinal injury, suggesting a correlation between these trophic factors and cortical remodeling. Yet, the inability to readily introduce a transgene or block endogenous genetic transcripts in the monkey brain has excluded this well-established model from molecular studies, restricting investigators to rodents, which are less amenable to studies of visual cortical function. Currently our laboratory has developed reliable methods to alter gene expression in the adult Macaca mulatta visual cortex using nonreplicative adeno-associated virus bearing a gene for enhanced green fluorescent protein (eGFP) providing persistent axon labeling and a gene encoding a small hairpin RNA designed to target and destroy selected neurotrophin messenger RNA by triggering the RNA interference pathway. Another challenge studying cortical plasticity in large animals is the lack of a multi- photon microscope capable of covering larger areas. Using a custom designed two-photon microscope for large animal imaging we can repeatedly image GFP labeled axons in the intact monkey brain, allowing us to longitudinally monitor real-time sprouting of horizontal connections in a single animal. Combining technical advances in viral gene delivery, real-time imaging and RNA interference we propose to inhibit the neurotrophin BDNF expression in vivo and determine if this factor is necessarily responsible for changes in the anatomical dynamics of horizontal connections after retinal lesions in the living transgenic monkey brain. ? ? ?
| Pena, John T G; Sohn-Lee, Cherin; Rouhanifard, Sara H et al. (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139-41 |
