Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons

Wickersham, Ian R.; Sullivan, Heather A.; Seung, H. Sebastian

doi:10.1038/nprot.2009.248

Cited by 145 publications

(195 citation statements)

References 36 publications

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“…EnvA-pseudotyped, G-deleted rabies viruses were produced in a manner similar to that described previously (27). (EnvA)SAD-ΔG-mCherry had functional titers of ∼2 × 10 10 infectious units per milliliter as determined through infection of TVA-expressing HEK293T cells.…”

Section: Methodsmentioning

confidence: 99%

Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

Wall

Wickersham

Çetin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

326

336

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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.ne of the most intractable problems in systems neuroscience has been the systematic description of neural connectivity in the intact mammalian brain. Many different types of neurons, each with distinct connectivity and function, can inhabit the same brain region. Even within a single neocortical column, dozens of types of projection neurons and local interneurons perform the computations that ultimately lead to the spiking output of that column. Through painstaking studies using molecular and cell biology techniques, electron microscopy, and electrophysiology, we are beginning to understand how a neuron's connectivity contributes to its function in the circuit in which it is embedded, but we lack efficient means for performing circuit-level analyses in vivo. Especially in brain regions with considerable neuronal heterogeneity, we are still greatly limited in our ability to study how groups of cells form their fine-scale connections, how these connections change over time, and how this plasticity affects a cell type's computational role in a dynamic circuit.Standard anterograde and retrograde neuronal tracers can reveal the locations of neurons projecting to or from particular brain regions, but fail to identify the cell types that actually receive the synaptic connections. This difficulty can be overcome by combining anterograde tracing with electron microscopy, but such studies are extremely tedious, often requiring years to elucidate just a small fraction of the possible connections. Furthermore, even EM studies do not readily reveal which cell types contribute to anterogradely labeled synapses or which cell types receive these connections. Over the last two decades, electrophysiological experiments in living brain slices have made it possible to more readily assay connections at high resolution. Although still relatively cumbersome, these studies have revealed that connectivity is extremely precise, allowing investigators to decipher many of the rules of local circuit connectivity. However, slice studies are limited in their ability to...

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Section: Methodsmentioning

confidence: 99%

Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

Wall

Wickersham

Çetin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

326

336

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“…For injections, we used a variant of the SAD-B19 vaccine strain of rabies virus designed by Wickersham and Wickersham et al (2010). In total, eight virus injections were made (see Fig.…”

Section: Rabies Virus Injectionsmentioning

confidence: 99%

Parallel feedback pathways in visual cortex of cats revealed through a modified rabies virus

Connolly

Hashemi-Nezhad

Lyon

2012

J of Comparative Neurology

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The visual cortex of cats is highly evolved. Analogously to the brains of primates, large numbers of visual areas are arranged hierarchically and can be parsed into separate dorsal and ventral streams for object recognition and visuospatial representation. Within early primate visual areas, V1 and V2, and to a lesser extent V3, the two streams are relatively segregated and relayed in parallel to higher order cortex, although there is some evidence suggesting an alignment of V2 and V3 to one stream over the other. For cats, there is no evidence of anatomical segregation in areas 18 and 19, the analogs to V2 and V3. However, previous work was only qualitative in nature. Here we re-examined the feedback connectivity patterns of areas 18/19 in quantitative detail. To accomplish this, we used a genetically modified rabies virus that acts as a retrograde tracer and fills neurons with fluorescent protein. After injections into area 19, many more neurons were labeled in putative ventral stream area 21a than in putative dorsal stream region posterolateral suprasylvian complex of areas (PLS), and the dendrites of neurons in 21a were significantly more complex. Conversely, area 18 injections labeled more neurons in PLS, and these were more complex than neurons in 21a. We infer from our results that area 19 in cat is more aligned to the ventral stream and area 18 to the dorsal stream. Based on the success of our approach, we suggest that this method could be applied to resolve similar issues related to primate V3.

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“…Three days before cell transfection, thaw a tube of 293T cells and plate the cells in a 15-cm plate as previously described (Barker 2005;Wickersham et al 2010). 2.…”

Section: Rescue From Cdnamentioning

confidence: 99%

“…• cSPBN-4GFP (Wickersham et al 2010) or other plasmid encoding a RV genome from which G has been deleted.…”

mentioning

confidence: 99%

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Rabies Viral Vectors for Monosynaptic Tracing and Targeted Transgene Expression in Neurons

Wickersham

Sullivan

2015

Cold Spring Harb Protoc

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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.

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Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons

Cited by 145 publications

References 36 publications

Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

Parallel feedback pathways in visual cortex of cats revealed through a modified rabies virus

Rabies Viral Vectors for Monosynaptic Tracing and Targeted Transgene Expression in Neurons

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