Presented by Juan Mo Study Questions • Why zebrafish transgenetic lines are very important for studying photoreceptor degeneration? • The synapses in inner plexiform layer and in outer plexiform layer? • ON bipolar cell & OFF bipolar cell? • What are the larval and adult phenotypes of pde6cw59 and Xops:mCFPq13 lines? • Light and dark preference test? • How does morpholino injection knock down gene expression? Zebrafish: outstanding model studying photoreceptor degenerations larvae adult • • Contains red, green, blue and UV cone cells Contains rods Advantages: •Ex utero rapid development •Optical transparency •Permeability to drugs •Ease of making transgenetic animal •The acute visual abilities of larvae pde6cw59 transgenic line cone degeneration death of rod (larvae) rod persistence (juvenile and adult) Xops:mCFPq13 transgenic line rod degeneration loss of cone (mammanlian) cone unaffected (zebrafish) Hypothesis • Rod photoreceptors protects from cone degeneration-induced retinal remodeling and restore visual responses in zebrafish http://what-when-how.com/neuroscience/visual-system-sensory-system-part-2/ metabotropic glutamate receptor ionotropic glutamate receptor Na+ K+ http://what-when-how.com/neuroscience/visual-system-sensory-system-part-2/ A B M ON ON OFF OFF ribbon http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/oute-plexiform/ Confocal Microscopy Result 1: development of ON bipolar cell contacts with rod and cone photoreceptors nyx:mYFPq16 cones rods OPL ON bipolar cell Cone pedicle ON bipolar cell Rod spherule dendrites cones rods Result 2: cone degeneration in pde6cw59 mutant larvae leads to ON bipolar cell remodeling pde6cw59 transgenic cone degeneration cones ON bipolar cell ON bipolar cell rods Xops:mCFPq13 transgenic ON bipolar cell rod degeneration ON bipolar cell Result 2: cone degeneration in pde6cw59 mutant larvae leads to ON bipolar cell remodeling pde6cw59 transgenic D6 ON bipolar axon terminals cone degeneration 6 DPF D8 Xops:mCFPq13 transgenic 8 DPF rod degeneration Result 3: postsynaptic glutamate receptor expression is altered in pde6cw59 larvae OFF bipolar cell ON bipolar cell horizontal cell ON bipolar cell D6 pde6cw59 transgenic Xops:mCFPq13 transgenic 6 DPF cone degeneration D8 rod degeneration 8 DPF Result 4: synaptic structure is maintained in Xops:mCFPq13 and pde6cw59 adults cones ON bipolar cell ON bipolar cell cones ON bipolar cell ON bipolar cell cones ON bipolar cell ON bipolar cell Result 4: synaptic structure is maintained in Xops:mCFPq13 and pde6cw59 adults photoreceptor terminals photoreceptor terminals photoreceptor terminals horizontal cell horizontal cell Cone pedicle rod spherule horizontal cell Result 5: visual responses are preserved in pde6cw59 mutant adults diffuse white light Result 5: visual responses are preserved in pde6cw59 mutant adults • Optokinetic response (OKR): http://www.youtube.com/watch?v=mfstlvsW1IY • WT and pde6cw59 adults display a robust OKR. Result 6: increased rod number protects the larval retina from cone degenerationinduced remodeling nuclear rod ON bipolar cell Morpholino: knock down gene translation Conclusion • Cone degeneration leads to ON bipolar cell remodeling in larvae. • Although the neural connectivity remains intact, the loss of the majority of the presynaptic input leads to significant remodeling observed in cone degeneration adult. • Visual responses are preserved in cone degeneration adult. • Increased rod number protects the larval retina from cone degeneration-induced remodeling. ? Rod photoreceptors protects from cone degenerationinduced retinal remodeling and restore visual responses in zebrafish Review Questions • Why zebrafish transgenetic lines are very important for studying photoreceptor degeneration? • The synapses in inner plexiform layer and in outer plexiform layer? • ON bipolar cell & OFF bipolar cell? • What are the larval and adult phenotypes of pde6cw59 and Xops:mCFPq13 lines? • Light and dark preference test? • How does morpholino injection knock down gene expression?