neurotechniques I.pptVIP

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * BDA Reiner et al. 2000 Anterograde tracing of corticospinal axons Biocytin/Neurobiotin Application: 5% solution, pressure injection or iontophoresis Fast degradation-short survival time 2-3 days Mostly anterograde transport Requires glutaraldehyde fixation Retrograde tracers All anterograde tracers are partially transported retrogradely Purely retrograde tracers: Fast Blue: UV dye accumulates in soma Diamidino Yellow: UV dye, accumulates in nucleus Microspheres: filled latex beads, accumulate in soma Fast Blue (FB) Diamidino Yellow (DiY) Application: 1-3 % aqueous solution Intensity stronger after longer transport times but leakage Microspheres Uptake mainly by terminals, uptake by fibers of passage?? Edmund Hollis, UCSD Scale bar 100 μm Latex or polystyrene beads filled with fluorophores Purely retrograde transport Cell morphology not well visible, granular appearance of labeled cells, permanent, non-toxic Fluorogold Application : 1-10%, pressure injection or iontophoresis Retrogradely transported Often granular appearance of labeled cell somata Antibodies against Fluorogold available Exc.: 325 nm, emm.:440 nm Labeling for extended time: several months Long-term toxicity Fluorogold Naumann et al. 2000 Fluorescence Immunolabeling Layer V Corticospinal neurons Ling Wang, UCSD Cell Filling with Lucifer Yellow Cell filling Ling Wang, UCSD Viruses Replication competent neurotropic viruses: HSV Pseudorabies Multisynaptic retrograde tracing Highly sensitive as viruses replicate after transsynaptic transport Pathways over several orders of synapses can be followed depending on the survival time Replication incompetent viruses expressing reporter genes: AAV/Lentivirus expressing GFP Choosing the Right Tracer Points to consider: Anterograde or retrograde tracing Transport time Efficient transport in investigated system: Age o