Can transsynaptic viral strategies be used to reveal functional aspects of neural circuitry?

https://doi.org/10.1016/j.jneumeth.2020.109005Get rights and content

Highlights

  • One-step RABV mapping does not recapitulate key aspects of functional connectivity.

  • Viruses label only a small fraction of the inputs onto first-order neurons.

  • The majority of RABV spread is likely synaptic, though non-synaptic spread occurs.

  • Users of transsynaptic technologies should be aware of limits in data interpretation.

  • A deeper understanding of how viruses transmit in the brain is required.

Abstract

Viruses have proved instrumental to elucidating neuronal connectivity relationships in a variety of organisms. Recent advances in genetic technologies have facilitated analysis of neurons directly connected to a defined starter population. These advances have also made viral transneuronal mapping available to the broader neuroscience community, where one-step rabies virus mapping has become routine. This method is commonly used to identify inputs onto defined cell populations, to demonstrate the quantitative proportion of inputs coming from specific brain regions, or to compare input patterns between two or more cell populations. Furthermore, the number of inputs labeled is often assumed to reflect the number of synaptic connections, and these viruses are commonly believed to label strong synapses more efficiently than weak synapses. While these maps are often interpreted to provide a quantitative estimate of the synaptic landscape onto starter cell populations, in fact very little is known about how transneuronal transmission takes place. We do not know how these viruses transmit between neurons, if they display biases in the cell types labeled, or even if transmission is synapse-specific. In this review, we discuss the experimental evidence against or in support of key concepts in viral tracing, focusing mostly on the use of one-step rabies input mapping and related methods. Does spread of these viruses occur specifically through synaptic connections, preferentially through synapses, or non-specifically? How efficient is viral transneuronal transmission, and is this efficiency equal in all cell types? And lastly, to what extent does viral labeling reflect functional connectivity?

Section snippets

Brief history of neural circuit mapping and development of one-step viral methods

The brain consists of a highly complex network of neurons which transmit information largely via synaptic connections. The understanding of how neurons are connected is thus critical to understanding both the basic and emergent properties of neural circuits. Though the advent of the microscope enabled an appreciation of the cellular structure of most tissues, the brain remained recalcitrant to study, in large part because thin sections yielded gray blobs of tissue that showed little about the

What is the evidence that viruses label synaptically-connected populations?

RABV and HSV/PRV are commonly assumed to spread through synaptic connections, and this assumption is rarely questioned. The original assertions about the transsynaptic preference of viral transmission can be traced back to an observational study by Ernest Goodpasture and Oscar Teague in 1923, where HSV was transferred from the lip of a human patient onto a rabbit’s cornea, where it transmitted via the optic nerve to the brain and produced encephalitis (Goodpasture and Teague, 1923). RABV and

How efficient is viral transmission? Do viruses label input cells equally?

An important consideration in any viral transneuronal experiment is the percentage of total input cells to the cell population of interest that get labeled by the virus. This number is difficult to discern when tracing inputs onto populations of starter neurons but is feasible when examining inputs onto single cells. The first study to provide this estimate was from Marshel and colleagues, who performed RABV one-step input tracing experiments from single layer 2/3 cortical neurons (Marshel et

To what extent does viral mapping reflect functional connectivity?

The premise of neuroanatomy is that an understanding of neural connectivity is necessary for understanding brain function. However, it is clear that even a detailed understanding of connectivity is not sufficient. An important limitation of viral tracing is that the extent to which viral labeling reflects functional aspects of neuronal connectivity has not been thoroughly explored. One significant barrier is that for most circuits in the brain, we lack a rigorously validated connectivity map of

Conclusion

In consideration of over a decade of RABV one-step tracing experiments from multiple cell types over many different circuits, there is no clear evidence that the extent of viral labeling can be used on its own to reproduce all aspects of functional connectivity. However, the majority of the data suggest that RABV input mapping can be used to detect synaptically-connected neurons, as where RABV labeling has been reported and tested, synaptic connections have also been observed, at least on a

CRediT authorship contribution statement

Alexandra Rogers: Writing - original draft, Writing - review & editing. Kevin T. Beier: Conceptualization, Visualization, Writing - original draft, Writing - review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

We have no competing interests to declare.

Acknowledgements

We would like to thank Michael Toledano and other members of the Beier lab for a critical review of this manuscript. This review was supported by the NIH (R00 DA041445, DP2 AG067666), Tobacco Related Disease Research Program (T31KT1437, T31IP1426), American Parkinson Disease Association (APDA-5589562), Alzheimer’s Association (AARG-NTF-20-685694), New Vision Research (CCAD2020-002), and the Brain and Behavior Research Foundation (NARSAD 26845).

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      The question of whether transmission of RABVs is strictly synapse specific is also under debate. It is therefore advisable to use RABV tracing for comparative and explorative studies rather than aiming for complete quantification of all monosynaptic inputs to cells (see Rogers and Beier [2021] for a comprehensive review of these limitations). This point becomes particularly pertinent when using Cre-dependent viruses because leaky expression of TVA can result in spurious rabies expression in cells that (combined with the staining issues described below) might be wrongly identified as input cells (Figure 2B; Miyamichi et al., 2013).

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