High-resolution neuroanatomical tract-tracing for the analysis of striatal microcircuits.
Although currently available retrograde tracers are useful tools for identifying striatal projection neurons, transported tracers often remained restricted within the neuronal somata and the thickest, main dendrites. Indeed, thin dendrites located far away from the cell soma as well as post-synaptic elements such as dendritic spines cannot be labeled unless performing intracellular injections. In this regard, the subsequent use of anterograde tracers for the labeling of striatal afferents often failed to unequivocally elucidate whether a given afferent makes true contacts with striatal projections neurons. Here we show that such a technical constraint can now be circumvented by retrograde tracing using rabies virus (RV). Immunofluorescence detection with a monoclonal antibody directed against the viral phosphoprotein resulted in a consistent Golgi-like labeling of striatal projection neurons, allowing clear visualization of small-size elements such as thin dendrites as well as dendritic spines. The combination of this retrograde tracing together with dual anterograde tracing of cortical and thalamic afferents has proven to be a useful tool for ascertaining striatal microcircuits. Indeed, by taking advantage of the trans-synaptic spread of RV, different subpopulations of local-circuit neurons modulating striatal efferent neurons can also be identified. At the striatal level, structures displaying labeling were visualized under the confocal laser-scanning microscope at high resolution. Once acquired, confocal stacks of images were firstly deconvoluted and then processed through 3D-volume rendering in order to unequivocally identify true contacts between pre-synaptic elements (axon terminals from cortical or thalamic sources) and post-synaptic elements (projection neurons and/or interneurons labeled with RV).
Developmental Biology Institute of Marseille-Luminy, UMR 6216 CNRS-Université de la Méditerranée, Marseille, France.
Pascal
P
Salin
María
M
Castle
Philippe
P
Kachidian
Pedro
P
Barroso-Chinea
Iciar P
IP
López
Alberto J
AJ
Rico
Lydia
L
Kerkerian-Le Goff
Patrice
P
Coulon
José L
JL
Lanciego
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001370:Axonal Transport / Q000502:physiology; D001931:Brain Mapping / Q000379:methods*; D003342:Corpus Striatum / Q000166:cytology* / Q000378:metabolism; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique / Q000379:methods; D007395:Interneurons / Q000166:cytology / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D009415:Nerve Net / Q000166:cytology* / Q000378:metabolism; D009434:Neural Pathways / Q000166:cytology / Q000378:metabolism; D009445:Neuroanatomy / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000378:metabolism / Q000821:virology; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D011820:Rabies virus / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling / Q000379:methods*; D009435:Synaptic Transmission / Q000502:physiology; D014764:Viral Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D014764:Viral Proteins