Hi, we injected an AAV vector containing an hM4Di DREADD in the VTA of newborn mice (aged P4/P5) using a protocol that was previously used in another lab. The mice had apparently a normal recovery and development the first three weeks, but after separating them from their mother and starting the behabioural experiments, many of them died. They were a bit smaller than usual, it seemed that they didn't drink or eat very much and suffered for hypothermia, all this seemed to be the cause of their death. We were wondering if the injection AAV injection could have caused some cell death, but didn't find any evidence yet.
Dear Ester,
How did you inject the vector? intraperitoneal?
Also did you use or injected any other chemicals, such as DMSO, with the vector?
best wishes, Refik
I injected the vector intracraneally, diluted in the comercial solution, which is supposed to be appropriate for the injection. We hadn't used DMSO when the mice died...
Dear Ester,
I have seen similar issues in the past from injections in the newborn mice. I don't know exactly what it is, but I have suspicion that it may be due infection as a result of the injection.
best wishes,
Refik
Hi Ester, did you check the extent of the infection? are you by chance creating a large region (that eventually can be traumatic for young pups) during injection ?
This could be an issue. In addition depending on the titer of the virus ... I would check whether there are some documented toxicity problems using the amount you have used (neurotoxicity I mean).
I would try to inject tiny amounts (20-50nL) very slowly (I know is not easy with such small mice because you can't use a stereotaxic frame). Try to keep the pups' head steady and with the aid of a manipulator maybe just try to be very slow in your injection.
Hi Ester,
AAV can absolutely kill neurons, especially if you have overly high titers. However, you probably would have see the effects of AAV induced neuronal death before weening. You mentioned that you inject AAV containing the inhibitory DREADD into the VTA.
Are you using a Cre/Flp activated DREADD into a Cre/Flp recombinase-VTA transgenic mouse? Or is the DREADD being globally expressed?
Did you start behavior testing (activating DREADD) at 3-4 weeks old?
If so, how did you administer the CNO (IP or drinking water)? CNO in drinking water is bitter to the taste, adding saccharin can help.
Assuming your DREADD is only inhibiting the VTA something to consider: The VTA projects to the nucleus accumbens and is involved in the reward pathway. By inhibiting the VTA, mice may loose the desire to eat or drink.
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