A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons

Abstract

We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “in vivo” transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs.

Individual mediodorsal thalamic neurons project to multiple areas of the rat prefrontal cortex: A single neuron‐tracing study using virus vectors

“…However, we could not determine how many types of thalamocortical projection patterns there were in the MD. Recently, a new method for axon labeling of long‐range projection neurons was reported (Porrero et al, ). The new method is based on in vivo transfection of the single‐strand palGFP–Sindbis RNA construct, and achieves more spatially precise and highly efficient single‐neuron labeling than the present method, injection of the Sindbis virus vectors.…”

Connectomic Analysis of Brain Networks: Novel Techniques and Future Directions

“…While modern technology is making the gathering of great amounts high-resolution data easier, these data would be of no use if it cannot be processed appropriately. Currently, along with simple and consistent single-cell labeling (Porrero et al, 2016) data analysis is in fact one of the two key limiting steps in connectome research (Helmstaedter, 2013): no automated projection tracing software is available that outperforms human annotators and hence most projection tracing is performed manually (but see also http://diademchallenge.org, where several algorithms can be found that were the result of an automated projection tracing-competition). Mapping all the myriad of highly diverse mouse brain neuronal networks and synapses with manual tracing may take thousands or even millions of hours.…”

“…In some cases, the viral vectors (e.g., retroviruses) can only infect mitotic neuronal precursors and thus can only be applied on embryonic brains. An alternative for directing the virus to a specific location is to inject or electroporate the vector RNA or DNA construct directly into the neuron in vivo (Porrero et al, 2016). …”

“…Imaging devices, no matter how sophisticated, will only achieve cellular resolution when dendrites, somas and axons have been sparsely, completely and specifically labeled (Porrero et al, 2016). …”

“…These include the intracellular or juxtacellular labeling with chemical markers such as biocytin or a variety of tagged dextrans (Pinault, 1996; Reiner et al, 2000), as well as transfection with viral vectors that drive the expression of high levels of fluorescent proteins in a few or even single cells (Kuramoto et al, 2009; Wang et al, 2014; Porrero et al, 2016). Alternative approaches rely on the generation of specific transgenic mice lines in whose brains high expression of label proteins is restricted (either by random insertion or through a promotor-specific Cre-lox system) to small neuron populations or a given cell type (Wouterlood et al, 2014; Saunders and Sabatini, 2015; see below).…”