Frequently Asked Questions: Neuronal Tracing Tool Service
1.What is Neurotropic virus?
2.What is the required biosafety level for using recombinant neurotropic viruses?
3.What are the recommended storage conditions of recombinant neurotropic viruses?
4.What are the cautions during virus use?
5.Whether the virus can be diluted?
6.What is the reason why the fluorescent signal is not detected after the virus injection?
7.What is the reason why the fluorescent signal is very weak after the virus injection?
8.How long can optogenetic and chemogenetic viruses be left in the brain after injection?
9.What is the injection volume and titer of the RV?
10.How long after RV injection can animals be sacrificed?
11.What percentage of the AAV helper is used in trans-monosynaptic system of RV?
12.How long after RV-EnvA-∆G-dsRed injection can animals be sacrificed?
13.What are the titer and volume of VSV when injecting animals?
14.How long after VSV injection can animals be sacrificed?
15.What are the titer and volume of HSV when injecting animals?
16.How long after HSV injection can animals be sacrificed?
17.What are the titer and volume of PRV when injecting animals?
18.How long after PRV injection can animals be sacrificed?
19.What range of nuclei can be infected with 200nLPRV injection?
20.What are the conditions for AAVs with serotype 1 for monosynaptic tracing?

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1.What is Neurotropic virus?
Neurotropic viruses are viruses that infect nerve cells and propagate in neural circuits, and it commonly include RV and VSV of the rhabdoviruses family and HSV-1-129 and PRV of the ɑ-herpesviridae family.
2.What is the required biosafety level for using recombinant neurotropic viruses?
When carrying out neural circuit tracing experiments with virus tracer, the researchers must handle the materials under Biosafety Level 2 (BSL-2).
3.What are the recommended storage conditions of recombinant neurotropic viruses?
4 ℃ for short-term storage and -80 ℃ for long-term storage. Each freeze-thaw will reduce 30 % of the titer, so repeated freeze-thaw cycles should be avoided.
4.What are the cautions during virus use?
Keep it in a 4℃ refrigerator or on ice during use, and minimize storage time at room temperature.
5.Whether the virus can be diluted?
Appropriate dilution can be performed with sterile PBS.
6.What is the reason why the fluorescent signal is not detected after the virus injection?
There are three common reasons:
● The needle tip may be blocked and the virus was not injected into the target area.
● Maybe the RV/HSV/VSV virus injection site does not overlap with helper virus in the trans-monosynaptic tracing system .
● Due to the improper storage or repeated freeze-thaw, the virus has lost its infectious activity.
7.What is the reason why the fluorescent signal is very weak after the virus injection? 
● Improper operation results in reduced infection activity.
● Maybe the RV/HSV/VSV virus injection site does not overlap with helper virus in the trans-monosynaptic tracing system .
8.How long can optogenetic and chemogenetic viruses be left in the brain after injection?
The signal expression is good in 2 months, and it starts to decay after 3 months, and the signal attenuation is more serious after 4 months. You can do behavioral tests within four months, but the longer you wait, the less attractive the signal gets. It's recommended that animals should be sacrificed within two months.
9.What is the injection volume and titer of the RV?
It's recommended to inject 100-300nL with the titer is 10^8IFU/mL and the injection speed is 20-50nL/min.
10.How long after RV injection can animals be sacrificed?
In general, the heart of animals could be irrigated one week later, and brain tissues could be taken after 4%PFA fixation to prepare samples for observation.
11.What percentage of the AAV helper is used in trans-monosynaptic system of RV?
It's recommended rAAV-DIO-GFP-TVA(serotype 9): rAAV-DIO-RVG(serotype 9) is 1:1 or 1:2 with the titer is 1-2.00E+12vg/ml. And the total volume is 200-300nl.
12.How long after RV-EnvA-∆G-dsRed injection can animals be sacrificed?
AAV-helper should be expressed for 2-3 weeks after injection to ensure high expression of both TVA and G proteins. RV-EnvA-∆G-dsRed is then injected at the same injection site, and the animal can be sacrificed after 7-12 days.
13.What are the titer and volume of VSV when injecting animals?
The titer is 10^9 IFU/mL for trans- multisynaptic and 10^7 IFU/mL for trans- monosynaptic. It's recommended to inject 50-300nL in CNS and 1-3ul in peripheral region.
14.How long after VSV injection can animals be sacrificed?
VSV is highly toxic. Animals can survive for 3-4 days after injecting in CNS and 5-8 days after peripheral injection. The survival time is different in animals with different constitution. According to experience, VSV needs 24-36h to across 1-2 levels in the central region, and 30-48h to across 2-3 levels, so appropriate time points can be selected according to the experiment.
15.What are the titer and volume of HSV when injecting animals?
It's recommended to inject 50-300nL in CNS and 1-3ul in peripheral region with the titer is 10^9 IFU/mL.
16.How long after HSV injection can animals be sacrificed?
HSV-1-129 is highly toxic. Animals can survive for 3-5 days after injecting in CNS and 5-9 days after peripheral injection. The survival time is different in animals with different constitution. According to experience, HSV needs 24-48h to across 1-2 levels in the central region, and 36-72h to across 2-3 levels, so appropriate time points can be selected according to the experiment. Also, checking the animal's status frequently to avoid premature death.
17.What are the titer and volume of PRV when injecting animals?
It's recommended to inject 50-300nL in CNS and 1-3ul in peripheral region with the titer is 10^9 IFU/mL.
18.How long after PRV injection can animals be sacrificed?
PRV is highly toxic. Animals can survive for 3-5 days after injecting in CNS and 5-9 days after peripheral injection. The survival time is different in animals with different constitution. According to experience, PRV needs 24-48h to across 1-2 levels in the central region, and 36-72h to across 2-3 levels, so appropriate time points can be selected according to the experiment. Also, checking the animal's status frequently to avoid premature death.
19.What range of nuclei can be infected with 200nLPRV injection?
The injection of 200nL virus forms a nearly spherical diffusion space in the brain area with a diameter of about 500nm.
20.What are the conditions for AAVs with serotype 1for monosynaptic tracing?
The ability of rAAV to cross synapses is low. rAAV2/1-CRE of 10^13vg/ml combined with rAAV2/9-DIO-eGFP virus can achieve anterograde monosynaptic tracing. If the rAAV2/1-CRE carries other components, it may be less efficient to achieve anterograde monosynaptic tracing.