Retrograde neuronal tracing with a deletion-mutant rabies virus

Abstract

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.A fundamental question regarding brain organization is how the various structures are connected to each other. A standard means of addressing it has been to inject into a region of interest some 'retrograde tracer': a substance taken up by the axon terminals of neurons that project to the injection site and which allows either visualization of their anatomy or, in the case of fluorescent tracers, their targeting for physiological study 1,2 . The rise of molecular biology has allowed the use of retrogradely infectious viruses expressing genetically encoded fluorophores 3,4 . In no published cases, however, has the resulting fluorescence been bright enough to consistently provide detailed anatomical information without immunohistochemical amplification and therefore to permit high-resolution identification of live labeled neurons for subsequent physiological study. Here we present a newly created virus that, because of several unique characteristics, does achieve these goals.Rabies virus, which has been used as a trans-synaptic tracer 5 , infects neurons through axon terminals and spreads between synaptically coupled neurons in an exclusively retrograde direction. In earlier work, K.K.C. and colleagues produced a virus that had the envelope glycoprotein gene deleted from its genome but that was grown in complementing cells so that the glycoprotein itself was incorporated into the viral particles'membranes despite the lack of its coding sequence in the viral genome 6 . Such a virus can infect contacted cells normally and, because the glycoprotein plays no role in transcription and replication, can still express its remaining genes and proliferate the viral core within initially infected cells. However, with no means of synthesizing glycoprotein in these cells, the newly created progeny are unable to infect other cells 6,7 , transforming the virus into a first-order retrograde tracer instead of a transsynaptic one. To reveal detailed morphology, we substituted the gene for enhanced green COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Methods website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ Note: Supplementary information is available on the Nature Methods website. (Fig. 1a). The result should be a virus that can infect initially contacted cells, replicate its core to high copy number and express high levels of EGFP, but be unable to spread beyond these initially infected cells. We termed this virus, which ...

Neuronal LRP4 regulates synapse formation in the developing CNS

“…As the experiments reported in our study affected LRP4 expression only in neurons (due to the neuron-specific synapsin promoter), the altered spine density that we observed must be caused by changes in neuronally expressed LRP4. Moreover, using a rabies virus-mediated monosynaptic tracing technique (Wickersham et al, 2007a), we demonstrated that the decreased number of synapse-like specializations and dendritic spines mediated by the expression of LRP4 miRNAs is paralleled by a decrease in the number of synaptic inputs as well, and therefore functional synapses. Hippocampal and cortical neurons in culture also responded to reduced LRP4 expression by changes in dendritic morphology.…”

“…Furthermore, changes in frequency of mEPSCs are not always proportional to the number of synapses, as changes in the stochastic release of presynaptic neurotransmitters or density of postsynaptic receptors could affect the mEPSC values as well. Thus, to further examine whether LRP4 expression levels affect synaptic connectivity, we used the rabies virus-mediated monosynaptic tracing technique, a method that allows an efficient analysis of functional presynaptic partners (Wickersham et al, 2007a). For that, cortical neurons were transfected with a retroviral vector encoding the EnvA-receptor TVA, the rabies virus glycoprotein G (which is responsible for retrograde transport of the virus across synapses), and the fluorescent reporter DsRedExpress2 (Fig.…”

“…5A). Neurons transfected with this vector are susceptible to subsequent infection by the glycoprotein G-deficient, eGFP-encoding EnvApseudotyped rabies virus (RABV) and capable of retrograde transfer of the virus to all immediate presynaptic partners (Deshpande et al, 2013;Wickersham et al, 2007a) (Fig. 5B).…”

“…The connectivity with presynaptic neurons was determined by counting the double-fluorescent cells (GFP + /DsRED + ) and the RABV-only single-positive cells (GFP + ). Results were expressed as connectivity ratio, representing the number of GFP-positive cells per GFPand DsRed-double positive cells as described (Wickersham et al, 2007a).…”

Selective Manipulation of Neural Circuits

“…Now, several serotypes of adeno-associated virus (AAV) such as AAV6, AAV8, and AAV9 are characterized to transduce neurons retrogradely [61][62][63][64][65], and lentivirus has been modified with its glycoprotein fused with rabies virus glycoprotein for retrograde transduction [41,42,66,67]. These viruses are easy to produce, result in less inflammation than herpes simplex and rabiesderived vectors, and have long duration and high level of transgene expression [68][69][70][71][72].…”