Perspectives in Demonstrating Anatomical Connectivity Labelling all Synaptic Connections of a Single Neuron

Labeling of "one neuron with a substance that would then clearly stain all the neurons connected to it, and no others" (Francis Crick) has been a sort of holy grail in the field of neural circuitry, and many attempts to achieve this have failed. Wickersham et al. (2007) have developed a strategy which uses a modified rabies virus whose genome is missing a gene required to make infectious viral particles. It would thus replicate within a neuron but spread no further because it is missing an envelope glycoprotein that it requires to leave the cell. They further modified the virus so that it would only be able to infect cells expressing a receptor for an avian virus called TVA. By expressing TVA, as well as the missing envelope gene, it was possible to initiate viral infection in that one cell and to produce infectious viral particles. By virtue of complementation of the missing gene, these viral particles then spread to and labelled all of the cells that were retrogradely connected to the first neuron. But then the virus could spread no further because the infected neurons did not express the missing envelope gene. Since the genetic modification of the rabies virus included also a sequence expressing Green Fluorescent Protein (GFP), the spread of the virus could be visualized under the fluorescence microscope. This method was applied to pyramidal cells in cortical slice cultures and is compatible with in vivo physiological assays that will allow the visual responses of single neurons to be directly correlated with their detailed connectivity. If successful this method will be invaluable for teasing apart the detailed circuitry of the brain.

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