Search

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them., (© 2024. The Author(s).)

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons in vivo., Competing Interests: Declaration of interests I.R.W. is a consultant for Monosynaptix, LLC, advising on the design of neuroscientific experiments., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Rabies viral and lentiviral vectors are very useful tools for neuroscientists, but high titer and purity are critical for in vivo applications. Here we present a protocol for concentration and purification of viral stocks by ultracentrifugation on a sucrose step gradient to remove impurities of both higher and lower densities than the virus itself, with sucrose removed by a subsequent pelleting step. The final stocks are concentrated in volume by a factor of up to 1000, with higher expected purity than is obtained following previously published protocols for preparing G-deleted rabies viral vectors., (© 2015 Cold Spring Harbor Laboratory Press.)

Deletion-mutant rabies viral (RV) vectors are powerful tools for neuroscience, allowing monosynaptic tracing of inputs to defined populations and rapid, high-level transgene expression in neurons targeted by multiple routes. High titers and high purity are critical for the successful use of RV vectors in vivo. Here we present a protocol for producing high-quality viral stocks that can be concentrated by ultracentrifugation for final titers in excess of 10(10) infectious units per milliliter., (© 2015 Cold Spring Harbor Laboratory Press.)

An important aspect of any neural circuit is the placement of its output synapses, at levels ranging from macroscopic to subcellular. The many new molecular tools for locating and manipulating synapses are limited by the viral vectors available for delivering them. Adeno-associated viruses are the best current means of labelling and manipulating axons and synapses, but they have never expressed more than one transgene highly enough to label fine axonal structure while also labelling or perturbing synapses. Their slow expression also makes them incompatible with retrograde and transsynaptic vectors, preventing powerful combinatorial experiments. Here we show that deletion-mutant rabies virus can be specifically targeted to cells local to an injection site, brightly labelling axons even when coexpressing two other transgenes. We demonstrate several novel capabilities: simultaneously labelling axons and presynaptic terminals, labelling both dendrites and postsynaptic densities, and simultaneously labelling a region's inputs and outputs using co-injected vectors.

We describe a powerful system for revealing the direct monosynaptic inputs to specific cell types in Cre-expressing transgenic mice through the use of Cre-dependent helper virus and a modified rabies virus. We generated helper viruses that target gene expression to Cre-expressing cells, allowing us to control initial rabies virus infection and subsequent monosynaptic retrograde spread. Investigators can use this system to elucidate the connections onto a desired cell type in a high-throughput manner, limited only by the availability of Cre mouse lines. This method allows for identification of circuits that would be extremely tedious or impossible to study with other methods and can be used to build subcircuit maps of inputs onto many different types of cells within the same brain region. Furthermore, by expressing various transgenes from the rabies genome, this system also has the potential to allow manipulation of targeted neuronal circuits without perturbing neighboring cells.

Understanding how neural circuits work requires a detailed knowledge of cellular-level connectivity. Our current understanding of neural circuitry is limited by the constraints of existing tools for transsynaptic tracing. Some of the most intractable problems are a lack of cellular specificity of uptake, transport across multiple synaptic steps conflating direct and indirect inputs, and poor labeling of minor inputs. We used a novel combination of transgenic mouse technology and a recently developed tracing system based on rabies virus (Wickersham et al., 2007a,b) to overcome all three constraints. Because the virus requires transgene expression for both initial infection and subsequent retrograde transsynaptic infection, we created several lines of mice that express these genes in defined cell types using the tetracycline-dependent transactivator system (Mansuy and Bujard, 2000). Fluorescent labeling from viral replication is thereby restricted to defined neuronal cell types and their direct monosynaptic inputs. Because viral replication does not depend on transgene expression, it provides robust amplification of signal in presynaptic neurons regardless of input strength. We injected virus into transgenic crosses expressing the viral transgenes in specific cell types of the hippocampus formation to demonstrate cell-specific infection and monosynaptic retrograde transport of virus, which strongly labels even minor inputs. Such neuron-specific transgenic complementation of recombinant rabies virus holds great promise for obtaining cellular-resolution wiring diagrams of the mammalian CNS.

Recombinant rabies viruses rendered replication-deficient by the deletion of their envelope glycoprotein gene are useful tools for neuroscientists, permitting (1) extraordinarily high transgene expression levels within neurons, (2) retrograde infection of projection neurons through their axon terminals, (3) targeted infection of genetically specified neurons and (4) monosynaptic tracing of neuronal inputs. Here we present a detailed protocol for the production of high-titer and high-purity viral stocks, from initial generation of infectious virus from cDNA through amplification on complementing cell lines, pseudotyping if desired, purification by ultracentrifugation and titering. The procedure requires 3-4 weeks to complete.

We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.

The enormous potential of modern molecular neuroanatomical tools lies in their ability to determine the precise connectivity of the neuronal cell types comprising the innate circuitry of the brain. We used transgenically targeted viral tracing to identify the monosynaptic inputs to the projection neurons of layer II of medial entorhinal cortex (MEC-LII) in mice. These neurons are not only major inputs to the hippocampus, the structure most clearly implicated in learning and memory, they also are "grid cells." Here we address the question of what kinds of inputs are specifically targeting these MEC-LII cells. Cell-specific infection of MEC-LII with recombinant rabies virus results in unambiguous labeling of monosynaptic inputs. Furthermore, ratios of labeled neurons in different regions are largely consistent between animals, suggesting that label reflects density of innervation. While the results mostly confirm prior anatomical work, they also reveal a novel major direct input to MEC-LII from hippocampal pyramidal neurons. Interestingly, the vast majority of these direct hippocampal inputs arise not from the major hippocampal subfields of CA1 and CA3, but from area CA2, a region that has historically been thought to merely be a transitional zone between CA3 and CA1. We confirmed this unexpected result using conventional tracing techniques in both rats and mice. [ABSTRACT FROM AUTHOR]

The generation of purposive movement by mammals involves coordinated activity in the corticospinal and corticostriatal systems, which are involved in different aspects of motor control. In the motor cortex, corticospinal and corticostriatal neurons are closely intermingled, raising the question of whether and how information flows intracortically within and across these two channels. To explore this, we developed an optogenetic technique based on retrograde transfection of neurons with deletion-mutant rabies virus encoding channelrhodopsin-2, and used this in conjunction with retrograde anatomical labeling to stimulate and record from identified projection neurons in mouse motor cortex. We also used paired recordings to measure unitary connections. Both corticospinal and callosally projecting corticostriatal neurons in layer 5B formed within-class (recurrent) connections, with higher connection probability among corticostriatal than among corticospinal neurons. In contrast, across-class connectivity was extraordinarily asymmetric, essentially unidirectional from corticostriatal to corticospinal. Corticostriatal neurons in layer 5A and corticocortical neurons (callosal projection neurons similar to corticostriatal neurons) similarly received a paucity of corticospinal input. Connections involving presynaptic corticostriatal neurons had greater synaptic depression, and those involving postsynaptic corticospinal neurons had faster decaying EPSPs. Consequently, the three connections displayed a diversity of dynamic properties reflecting the different combinations of presynaptic and postsynaptic projection neurons. Collectively, these findings delineate a four-way specialized excitatory microcircuit formed by corticospinal and corticostriatal neurons. The "rectifying" corticostriatal-to-corticospinal connectivity implies a hierarchical organization and functional compartmentalization of corticospinal activity via unidirectional signaling from higher-order (corticostriatal) to lower-order (corticospinal) output neurons. [ABSTRACT FROM AUTHOR]

Understanding how neural circuits work requires a detailed knowledge of cellular-level connectivity. Our current understanding of neural circuitry is limited by the constraints of existing tools for transsynaptic tracing. Some of the most intractable problems are a lack of cellular specificity of uptake, transport across multiple synaptic steps conflating direct and indirect inputs, and poor labeling of minor inputs. We used a novel combination of transgenic mouse technology and a recently developed tracing system based on rabies virus (Wickersham et al., 2007a,b) to overcome all three constraints. Because the virus requires transgene expression for both initial infection and subsequent retrograde transsynaptic infection, we created several lines of mice that express these genes in defined cell types using the tetracycline-dependent transactivator system (Mansuy and Bujard, 2000). Fluorescent labeling from viral replication is thereby restricted to defined neuronal cell types and their direct monosynaptic inputs. Because viral replication does not depend on transgene expression, it provides robust amplification of signal in presynaptic neurons regardless of input strength. We injected virus into transgenic crosses expressing the viral transgenes in specific cell types of the hippocampus formation to demonstrate cell-specific infection and monosynaptic retrograde transport of virus, which strongly labels even minor inputs. Such neuron-specific transgenic complementation of recombinant rabies virus holds great promise for obtaining cellular-resolution wiring diagrams of the mammalian CNS. [ABSTRACT FROM AUTHOR]

Summary: There has never been a wholesale way of identifying neurons that are monosynaptically connected either to some other cell group or, especially, to a single cell. The best available tools, transsynaptic tracers, are unable to distinguish weak direct connections from strong indirect ones. Furthermore, no tracer has proven potent enough to label any connected neurons whatsoever when starting from a single cell. Here we present a transsynaptic tracer that crosses only one synaptic step, unambiguously identifying cells directly presynaptic to the starting population. Based on rabies virus, it is genetically targetable, allows high-level expression of any gene of interest in the synaptically coupled neurons, and robustly labels connections made to single cells. This technology should enable a far more detailed understanding of neural connectivity than has previously been possible. [Copyright &y& Elsevier]

• A guide to successfully conducting monosynaptic tracing experiments. • Instructions for constructing custom injection apparatus. • Recommendations for reagent preparation. • Detailed step-by-step surgical protocol. • Discussion of experimental design and necessary controls. Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (ΔG) rabies viral vectors. [ABSTRACT FROM AUTHOR]