Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In Vivo

Abstract

SUMMARY To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a novel strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately-labeled distal cortical and subcortical inputs. The broad applicability of this technique to probe and manipulate single neuronal networks with single-cell resolution in vivo may shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors

“…This rule is occasionally broken, however, as in the case of the infection of RMS cells. In addition, Muller glial infection has been shown for PRV (42), and RABV has been shown to sometimes infect cortical glia (43). VSV(LCMV-G), PRV, and RABV are thus not exclusively transsynaptic.…”

The brain parenchyma has a type I interferon response that can limit virus spread

“…Neurotropic viruses are increasingly used as transsynaptic tracers (22) but only label a fraction of the connections of infected neurons (23,24). The data presented here raise the possibility that innate immunity is at least one factor that contributes to this inefficiency.…”

Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb

“…Providing these starter cells with the RABV glycoprotein (G) allows for the subsequent retrograde transsynaptic virus transfer to presynaptic partners. By means of single-cell electroporation, adenoassociated virus-mediated transduction or Cre-mediated recombination in transgenic mice, previous strategies have proved the efficient expression of TVA and G in restricted populations of starter cells in vivo (11,12). The delivery of RABV to and subsequent transfer from these starter cells located in a host of neuroanatomical regions, such as the cerebral cortex and the Significance New neurons are constantly added to the hippocampus and the olfactory bulb.…”