Transneuronal circuit tracing with neurotropic viruses

The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Current opinion in neurobiology (Impact Factor: 6.63). 05/2009; 18(6):617-23. DOI: 10.1016/j.conb.2009.03.007
Source: PubMed


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.

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    • "More recently , the use of retrogradely infectious viruses expressing genetically encoded fluorophores has been developed through which details of neuronal morphology can be clearly labeled without immunohistochemical amplification. Recombinant rabies virus has been widely used to reveal detailed neuronal morphology (Callaway, 2008;Collo et al., 2014;Ugolini, 2011;Weible et al., 2010;Wickersham et al., 2007). In this study, we used this recombinant rabies virus-based method to reveal the dendritic morphology of dPSNs that projected their axons from low thoracic spinal cord to the upper lumbar cord level in adult rats. "
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    ABSTRACT: After spinal cord injury (SCI), poor regeneration of damaged axons of the central nervous system (CNS) causes limited functional recovery. This limited spontaneous functional recovery has been attributed, to a large extent, to the plasticity of propriospinal neurons, especially the descending propriospinal neurons (dPSNs). Compared with the supraspinal counterparts, dPSNs have displayed significantly greater regenerative capacity, which can be further enhanced by glial cell line-derived neurotrophic factor (GDNF). In the present study, we applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of dPSNs. We also investigated the neurotransmitters expressed by dPSNs after labeling with a retrograde tracer Fluoro-Gold (FG). dPSNs were examined in animals with sham injuries or complete spinal transections with or without GDNF treatment. Bilateral injections of G-Rabies and FG were made into the 2nd lumbar (L2) spinal cord at 3days prior to a spinal cord transection performed at the 11th thoracic level (T11). The lesion gap was filled with Gelfoam containing either saline or GDNF in the injury groups. Four days post-injury, the rats were sacrificed for analysis. For those animals receiving G-rabies injection, the GFP signal in the T7-9 spinal cord was visualized via 2-photon microscopy. Dendritic morphology from stack images was traced and analyzed using a Neurolucida software. We found that dPSNs in sham injured animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution with dorsal-ventral retraction and lateral-medial extension. Treatment with GDNF significantly increased the terminal dendritic length of dPSNs. The density of spine-like structures was increased after injury, and treatment with GDNF enhanced this effect. For the group receiving FG injections, immunohistochemistry for glutamate, choline acetyltransferase (ChAT), glycine, and GABA was performed in the T7-9 spinal cord. We show that the majority of FG retrogradely-labeled dPSNs were located in the Rexed Lamina VII. Over 90% of FG-labeled neurons were glutamatergic, with the other three neurotransmitters contributing less than 10% of the total. To our knowledge this is the first report describing the morphologic characteristics of dPSNs and their neurotransmitter expressions, as well as the dendritic response of dPSNs after transection injury and GDNF treatment.
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    • "The dual-virus approach described here capitalizes upon the high efficiency and longterm tolerability of AAVs while also enabling robust optical control (Figures 2D and 2E) via catalytically driven activation of high copy-number transgenes. It is important to be aware that direct transduction of axon terminals with certain viruses can be achieved for targeting (reviewed in Callaway, 2008), and indeed virtually all viruses (including AAVs) carry some degree of this capability in a serotype-and circuit-dependent manner, as we and others have observed (Burger et al., 2004; Paterna et al., 2004; Callaway, 2008; Lima et al., 2009; Passini et al., 2005; Nathanson et al., 2009). It is also important to consider (in the process of selecting viruses) the need for high levels of infection and expression while avoiding adverse effects on transduced neurons over weeks to months for long-term electrophysiological or behavioral experiments. "
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    DESCRIPTION: Molecular and Cellular Approaches for Diversifying and Extending Optogenetics (Viviana Gradinaru,Feng Zhang) university of california , los anjeles and caltech
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    • "Lentivirus provides several advantages with spatially defined expression and combinations of tracers with optoge - netic and other functional constructs allowing examination of questions not previously possible using traditional tracing techniques . Analysis of neuronal circuitry has traditionally relied on neuronal tracers such as biotinylated dextran amine ( BDA ) , phaseolus vulgaris leucoagglutinin ( PHAL ) , wheat - germ agglutinin ( WGA ) , horseradish peroxidase ( HRP ) , cholera toxin , and more recently pseudorabies virus to label neuronal pathways ( Callaway , 2008 ; Huh et al . , 2010 ) . "
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    ABSTRACT: Lentiviruses have been extensively used as gene delivery vectors since the mid-1990s. Usually derived from the human immunodeficiency virus genome, they mediate efficient gene transfer to non-dividing cells, including neurons and glia in the adult mammalian brain. In addition, integration of the recombinant lentiviral construct into the host genome provides permanent expression, including the progeny of dividing neural precursors. In this review, we describe targeted vectors with modified envelope glycoproteins and expression of transgenes under the regulation of cell-selective and inducible promoters. This technology has broad utility to address fundamental questions in neuroscience and we outline how this has been used in rodents and primates. Combining viral tract tracing with immunohistochemistry and confocal or electron microscopy, lentiviral vectors provide a tool to selectively label and trace specific neuronal populations at gross or ultrastructural levels. Additionally, new generation optogenetic technologies can be readily utilized to analyze neuronal circuit and gene functions in the mature mammalian brain. Examples of these applications, limitations of current systems and prospects for future developments to enhance neuroscience knowledge will be reviewed. Finally, we will discuss how these vectors may be translated from gene therapy trials into the clinical setting.
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