A light microscope-based double retrograde tracer strategy to chart central neuronal connections.
This protocol describes a double retrograde tracing method to chart divergent projections in the CNS using light microscope techniques. It is based on immunohistochemical visualization of retrograde transport of cholera toxin b-subunit (CTb) and silver enhancement of a gold-lectin conjugate. Production of the gold-lectin is explained in detail, and a technique is offered to record through the injection pipettes, to help guide accurate placement of injections. Visualization of the two tracers results in light brown staining of CTb-labeled neurons and labeling by black particles of gold-lectin-containing neurons. Both types of label are easily recognized in the same neuron. The labeling is permanent and is well suited for studies in which large areas of the brain need to be surveyed. The whole procedure (excluding survival time) takes approximately 5-7 d to complete.
Ruigrok
Tom J H
TJ
Department of Neuroscience, Erasmus MC Rotterdam, Rotterdam, The Netherlands. t.ruigrok@erasmusmc.nl
Apps
Richard
R
D000818:Animals; D001370:Axonal Transport / Q000502:physiology; D002415:Cats; D002490:Central Nervous System / Q000166:cytology*; D002772:Cholera Toxin / Q000032:analysis; D004553:Electric Conductivity; D006046:Gold / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D007267:Injections / Q000379:methods; D037102:Lectins / Q000032:analysis; D051379:Mice; D008853:Microscopy; D009434:Neural Pathways / Q000166:cytology*; D011689:Purkinje Cells / Q000502:physiology; D011817:Rabbits; D051381:Rats; D016622:Silver Staining; D009435:Synaptic Transmission
D037102:Lectins; D006046:Gold; D002772:Cholera Toxin