Transneuronal circuit tracing with neurotropic viruses

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Because neurotropic viruses naturally traverse neural pathways, they are extremely valuable for elucidating neural circuits. Naturally occurring herpes and rabies viruses have been used for transneuronal circuit tracing for decades. Depending on the type of virus and strain, virus can travel preferentially in the anterograde or the retrograde direction. More recently, genetic modifications have allowed for many improvements. These include: reduced pathogenicity; addition of marker genes; control of synaptic spread; pseudotyping for infection of selected cells; addition of ancillary genetic elements for combining circuit tracing with manipulation of activity or functional assays. These modifications, along with the likelihood of future developments, suggest that neurotropic viruses will be increasingly important and effective tools for future studies of neural circuits.

Introduction

A major goal of systems neuroscience is to understand how neural circuits generate perception and behavior. To this end, considerable effort has been devoted to revealing brain circuits. Such studies are most useful when they also allow links to be made to function, either by correlating activity with circuitry or by manipulating circuit components and monitoring changes in function or behavior. Such efforts are difficult, however, owing to the extreme complexity of the brain. For example, conventional anterograde and retrograde neuronal tracers have allowed the collection of extensive information about connectivity between visual cortical areas in the macaque monkey [1]. These data reveal what areas are directly connected, the locations of the cells that provide the connections, and the laminar zones of termination of afferent axons. This information has been used to guide studies in which likely functional interactions between areas are inferred and tested with lesions or electrical stimulation [2] [3]. But conventional tracers do not have sufficient resolution to reveal connectivity or guide functional studies at finer levels of complexity. For example, they do not reveal whether axons that terminate in a particular location do, or do not, make synaptic contacts onto particular cell types or onto cells that in turn connect to other cells. Instead, methods are required that can reveal multisynaptic pathways and can also identify connections to and from particular cell types. Ideally, such methods can also be integrated with functional studies.

There are many approaches that have been successfully exploited to reveal connectivity at high resolution [4]. Each of these has advantages and limitations. The purposes of this review are to highlight the advantages and limitations of neurotropic viruses; to provide sufficient basic information about the properties and behavior of neurotropic viruses to allow an understanding of the sources of these advantages and limitations; and to review recent progress in the exploitation of this understanding to allow the development of novel tracing systems that are based on neurotropic viruses. The advantages and limitations of other methods will not be directly addressed.

Neurotropic viruses have two main advantages that have been exploited in studies of neural circuits. These are the ability to traverse multisynaptic pathways and the ability of viral replication to amplify signals at each step in the process [5, 6, 7]. Furthermore, depending on the species and strain of the virus, viral spread can be highly specific to synaptically connected partners. Limitations also depend on the species and strain of virus and include a lack of synaptic specificity of spread, inability to target infection to particular cell types, dependence of the speed of spread on both strength and numbers of connections, and induction of cell death. More recently, the fact that viruses are genetic machines has been exploited to eliminate limitations of naturally occurring neurotropic viruses. Genetic modifications allow control over which cells are initially infected, control over the extent of viral spread, and control of the direction of spread. It is also possible to express useful genes directly from the viral genome.

Section snippets

Basic properties of neurotropic viruses

In order to understand the advantages and limitations of neurotropic viruses for tracing neural circuits, it is informative to first understand their basic properties and the similarities and differences between viral species and strains. Although there are numerous neurotropic viruses, only two major classes have typically been used to trace neural circuits experimentally, and these will be the focus of this review. These are herpes viruses and rabies virus. (It is important to note that one

Selectivity and reliability of viral spread

One of the most important characteristics to consider for any transneuronal tracer, including neurotropic viruses, is whether spread is restricted to neurons that are connected by synaptic contacts. Because the transfer of information between neurons is primarily dependent on synapses (and gap junctions), the most relevant circuit diagram is based exclusively on connectivity, not proximity. This issue is not fully resolved but has been most carefully explored for the PRV Bartha strain of herpes

Direction of viral spread and other differences between viruses and strains

The virulence and direction of transneuronal spread of neurotropic viruses depend both on the type of virus and the strain. Rabies virus spreads exclusively in the retrograde direction, from postsynaptic to presynaptic cells, regardless of the strain [6, 23]. Different strains of rabies virus do differ, however, in their rate of spread and their safety when used in a laboratory setting. Because the rate of spread of rabies virus can vary dramatically depending not only on strain but also on

Tracing connections between specific cell types and controlling the extent of spread

Owing to the complexity of the nervous system, it is extremely valuable to be able to trace connections to or from particular cell types. To some degree, neurotropic viruses make this possible simply by virtue of the fact that they can spread across multiple synaptic steps. For example, if a virus that spreads retrogradely is injected into area A, the initial infection will be restricted to cells in area A and to cell types in other areas, say area B, that project axons to area A. As the virus

Expression of additional gene products

A final advantage of genetically modified herpes and rabies viruses is that essentially any gene of interest can be inserted into the genome, so that it is expressed in selected cells with known connectivity. The most straightforward gene products are those to mark cells, such as fluorescent proteins. Since these come in many colors [41], it is possible to selectively mark various neuronal populations that have been labeled with different viruses. But these strategies can be extended to the

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Work in the author's laboratory is supported by the National Institutes of Health. The author is grateful to current and former members of his lab and, particularly, Ian Wickersham for numerous discussions and insights.

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