1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School
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Beier, K., Cepko, C. Viral Tracing of Genetically Defined Neural Circuitry. J. Vis. Exp. (68), e4253, doi:10.3791/4253 (2012).
Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).
1. Making Virus from cDNA: Recovery of VSV from cDNA using Vaccinia-T7 System10
2. Passage and Concentration of VSV
3. Virus Injection
4. Harvesting Tissue/Tissue Preparation
5. Representative Results
The virus that is rescued from cDNA should be able to infect TVA800 cells, but not 293T cells (Figure 3). Supplying the RABV-G permits the infection of multiple cell types, including 293T. However, since the A/RG genome is encoded in the genome, spread among cells occurs at a much higher rate in TVA-expressing cells.
Once the virus is concentrated, typical titers range between 108 ffu/ml and 1010 ffu/ml. These titers are adequate for use in vivo.
During LGN injection into the mouse, we do not distinguish dorsal LGN (dLGN) from ventral LGN (vLGN), as both typically become infected. Therefore, multiple RGC types become labeled (Figure 4), including those that project to the vLGN, such as melanopsin RGCs (Figure 4D) and ON-DSGCs (Figure 4E), and those that project to the dLGN, such as the small-arbor RGCs (Figure 4C) and ON-OFF-DSGCs (Figure 4F). Although more than one type of RGC transmitted virus to SACs, as reported elsewhere (Beier et al., in review), here we focus only on the well-studied connections of ON-OFF-DSGCs to SACs.
When we inject mice of the genotype cTVA(+)/ChAT-Cre(-), where no TVA should be expressed, only RGCs are labeled (i.e. Figure 4). These are due to initial uptake of the RABV-G pseudotyped virus by the RGCs, and no spread occurs. No RGCs are infected from an LGN infection of rVSV(A/RG) virus not pseudotyped with RABV-G, due to an inability of these virions to transverse the long axons of these RGCs. However, when injecting cTVA(+)/ChATCre(+) (and R26TdT(+)) mice into the LGN, viral spread does occur, only to red cells, which express the Cre reporter (i.e. Figure 5). These cells also co-label with the anti-ChAT antibody, and stratify only in the two ChAT laminae, confirming their identity as SACs (Figures 5B', C'). The number of virally labeled SACs per DSGC ranged from one to nine.
Figure 1. Our retrograde transsynaptic tracing system compared to the originally developed method. (A) (i) In the method developed by Wickersham and colleagues, "starter cells" are transfected with three genes: the TVA receptor, used to permit infection specifically of transfected cells; the RABV-G, used to complement the RABV, which itself had the G gene deleted; and a red fluorescent protein to identify transfected cells. (ii) A RABV pseudotyped with the A/RG protein infects TVA-expressing cells, making them yellow. (iii) Retrograde transsynaptic transmission occurs to upstream neurons. (B) (i) In our method, TVA-expressing cells are defined genetically, and are labeled with a conditional red fluorescent protein. (ii) The "starter cells" are those infected by a VSV encoding the A/RG gene in the viral genome. These starter cells do not express the TVA receptor. (iii) Retrograde transsynaptic transmission occurs to TVA-expressing neurons only if these cells provide synaptic input onto the starter neurons. Click here to view larger figure.
Figure 2. Schematic of the experimental procedure. (A) First, the virus is rescued from cDNA. It is then passaged and pseudotyped with RABV-G, and concentrated and titered. This virus is then injected into the brain, where it is allowed to incubate for the desired period of time. After this time, the eye is harvested, and the retina dissected and analyzed. (B) Schematic of pseudotyping the virus with RABV-G. This is necessary for infection of retinal ganglion cells from a brain injection. The RABV-G gene is first transfected into tissue culture cells expressing the TVA receptor. These cells are then infected with the rVSV(A/RG) virus. The virions released will have RABV-G in the viral envelope, but will not have the gene for RABV-G in the viral genome. (C) A schematic of the mouse crosses necessary for triple transgenic animals. Conditional TVA and tdTomato alleles are crossed to a Cre driver of choice, such that both TVA and tdTomato are expressed only in cells with a Cre expression history. In this example, we used ChAT-Cre. (D) A schematic of the retinal infection in order to trace DSGC-SAC circuits. The ChAT cells are made to express TVA and tdTomato, as shown in (C). RGCs are infected from an LGN infection of the virus produced in (B). As the virus expresses GFP, these RGCs will be green. The virus will then spread only to TVA-expressing ChAT amacrine cells if they are connected to the labeled RGC. These cells will be both red and green, or yellow. Click here to view larger figure.
Figure 3. Behavior of rVSV(A/RG) virus on 293T and TVA800 cells. These cells were infected with a low titer rVSV(A/RG) pseudotyped with RABV-G. The virus spreads between TVA-expressing cells in a matter of hours, as shown at 6 hours post infection (HPI), 1 and 2 days post infection (DPI). However, the spread on 293T cells, which do not express the TVA receptor, though it does occur, happens much slower. Scale bar = 100 μm.
Figure 4. Representative infections of RGCs of non-TVA expressing mice. Animals were injected into the LGN with the rVSV(A/RG) virus pseudotyped with RABV-G, and tissue harvested 2 dpi. (A) Sparse (RGCs indicated by yellow arrowheads) or (B) dense infections can be obtained, depending on the titer of virus and quality of injection. Many different types of RGCs can be identified based on morphology, including (C) small arbor RGCs, (D) type 1 melanopsin RGCs, (E) ON-DSGCs, and (F) ON-OFF-DSGCs, among others. Scale bars = 50 μm.
Figure 5. rVSV(A/RG) transmission from ON-OFF-DSGCs to SACs follows the expected pattern. (A) ON-OFF-DSGCs (white arrowhead with green fill) transmit virus to multiple SACs (yellow arrowheads). (A'). All green cells that are not the DSGC co-label with the Cre reporter, indicating that they are TVA-expressing SACs. (B-C) The DSGC (white arrowhead with green fill) and SACs (yellow arrowheads) stratify in the appropriate ChAT layers of the retinal inner plexiform layer (IPL). The cells to which the virus transmits include both ON and OFF-SACs. Numbers in panels B' and C' indicate the IPL laminae. The location of the DSGC soma is only indicated by the arrowhead but not shown in panel C to highlight the SACs. Scale bars = 50 μm.
Using viruses to study neural circuits is a relatively high throughput method of analyzing connected neurons. However, generating both VSV and RABV virions is not trivial. Although the protocol listed above for rescuing virus from cDNA is provided, it is still a low-probability event. The levels of each of the N, P, and L plasmids need to be finely adjusted, and many trials and replicates need to be done to ensure viral rescue. The formation of the ribonucleotide particle incorporating a full VSV genome RNA is a low-probability event, and therefore may take several attempts. This technique may require time for optimization, however, once it is optimized, rescuing viruses is much easier.
Analysis of viral transmission from individual RGCs is best done when RGC dendritic arbors do not overlap (i.e. Figure 4A). The amount of virus injected and injection coordinates can be adjusted for optimal infection. Also, the location of injection will affect the numbers and types of RGCs labeled (i.e. superior colliculus or superchiasmatic nucleus will label different numbers and types of RGCs than an LGN injection).
VSV has a number of advantages relative to PRV and RABV. The expression level of VSV is very rapid, being visible within hours9,15. This permits shorter incubation times. In this experiment, the A/RG glycoprotein was encoded within the viral genome, making the virus replication-competent. While making replication-competent rabies virus makes a BL3 containment level necessary, for VSV, it is only BL2. The glycoprotein could have been provided in trans in the RGCs, i.e. by prior infection of an AAV encoding the A/RG fusion protein. However, this strategy relies on double infection, and the levels of glycoprotein expression may not be optimal. When expressed from the viral genome, the glycoprotein levels are finely tuned as in the case of wild-type VSV. Without the need for co-infection, all RGCs labeled have the potential for viral transmission. The major drawback of VSV is its relatively rapid cell toxicity. We have examined VSV-infected cells by physiology, and noted that they were physiologically normal by 18 hr post-infection, but were too sick to obtain physiological measurements by 48 hr post-infection. Generally, neurons look morphologically normal for 2-3 days post-infection, but begin to show signs of sickness (blebbing) shortly thereafter. This is after the time in which transsynaptic transmission can be observed (24 hr). However, all transsynaptic viruses, including RABV and PRV, are toxic to the cells. Therefore, VSV is at present best used for an anatomical labeling tool, though we are in the process of making it better suited for physiological manipulations.
VSV is a particularly powerful transsynaptic neuronal tracer. By nature of its ability to accept different glycoproteins, it can trace neurons in the anterograde or retrograde directions9. Here, we show that it can also be used to probe a different question - whether or not a genetically-defined cell type inputs onto a starter cell population. Here we demonstrate its utility in the retina, however we also have used it to label genetically defined interneuronal populations the brain, with equal success. It can also be modified to contain a different glycoprotein, to permit transsynaptic anterograde transmission to TVA-expressing cells. The possibility of further manipulations offers the potential to further help analyze microcircuits and decode the complex computations of neural connections.
No conflicts of interest declared.
We would like to acknowledge Sean Whelan for assistance with rescuing recombinant VSV variants, and Didem Goz and Ryan Chrenek for technical assistance. This work was supported by HHMI (CLC), and #NS068012-01 (KTB).
|Baby Hamster Kidney (BSRT7) cells||available upon request|
|vaccinia (vTF7-3)||available upon request|
|pN, pP, pl plasmids||available upon request|
|HEK 293T cells||Open Biosystems||HCL4517|
|60 mm TC-Treated Culture Dish||Corning||430166|
|75 cm2 Rectangular Canted Neck Cell Culture Flask with Vent Cap||Corning||430641|
|Media : DMEM (Dulbecco’s Modified Eagle Medium)||Invitrogen||12491-015|
|1 M HEPES pH 7.4||Gibo||15630-080|
|FBS: Fetal Bovine Serum||Gibco||10437-028|
|Lipofectamine 2,000 Transfection Reagent||Invitrogen||11668-019|
|Syringe: 5 ml Luer-Lock syringe||Sigma||Z248010-1PAK|
|PEI: High Potency Linear PEI||Polysciences||23966|
|Corning 150 ml Tube Top Vacuum Filter System, 0.45 μm Pore||Corning||430314|
|Thinwall, Ultra-Clear, 38.5 ml, 25 x 89 mm ultracentrifuge tubes||Beckman-Coulter||344058|
|SW28 Ultracentrifuge rotor||Beckman-Coulter||342207|
|Ump injector||World Precision Instruments||Sys-Micro4|
|Four channel microcontroller||World Precision Instruments||UMP3|
|M.TXB Bench Motor with C.EMX-1 Dial Control, 115 Volt||Foredom||M.TXB-EM|
|H.10 Handpiece, Quick Change||Foredom||H.10|
|Step Drill, 0.5 mm||Foredom||A-58005P|
|Microelectrode holder||World Precision Instruments||MEH2S|
|1 ml syringe||Becton-Dickinson||309628|
|30 gauge injection needle||Becton-Dickinson||305106|
|Protective Ophthalmic Ointment||Doctors Foster and Smith||9N-014748|
|Surgery and Dissection tools|
|Scissors||Fine Science Tools||91402-12|
|Standard Forceps||Fine Science Tools||11000-12|
|Fine Forceps||Fine Science Tools||11255-20|
|Vannas spring scissors||Fine Science Tools||15000-00|
|Scalpel handle||Fine Science Tools||10003-12|
|Scalpel blades||Fine Science Tools||10015-00|
|Dissection and antibody staining|
|Phosphate Buffered Saline||Sigma||P4417|
|Donkey Serum||Jackson Immunoresearch||017-000-121|
|Donkey anti-chicken Dylight 488||Jackson immunoresearch||703-545-155|
|Donkey anti-chicken Alexa Fluor 647||Jackson immunoresearch||705-605-147|
|Superfrost plus microscope slides||Fisher||12-550-100|
|Cover glass 22 x 22, 0 thickness||Electron Microscopy Sciences||72198-10|
|Silicone elastomer||Rogers Corp||HT-6220|
|Clear nail polish||Electron Microscopy Sciences||72180|
|Prolong Gold antifade reagent||Invitrogen||P36930|