I am planning an experiment that utilizes 'targeted recombination in active populations' (TRAP) and involves stereotactically injecting the following viral cocktail in the PFC of BL/6 mice:
[AAV-cFos-ERT2-Cre-ERT2-PEST-no WPRE]
[pAAV-hSyn-DIO-hM3D(Gq)-mCherry]
After 3-4 weeks, mice will be injected with Tamoxifen, followed by stimulation with alogenes (23-24 hrs after TM injection). The cFos-positive cells in PFC are assumed to express hM3D(Gq) in a Cre- and TM-dependent manner after stimulation. These neurons can then be exclusively activated by hM3D(Gq) ligands, like CNO.
Issue
I have a question regarding the time it takes for hM3Dq to be expressed after TRAPing. I would like to know the time course of events that occur after stimulation. Any technical articles, protocols, or books that may provide more information are very welcome!
The information in the articles I have come across seems to be controversial.
For example, Xuetao Qi et al. (2022) used a similar but not the same genetic strategy (tTa-Dox) to gain access to nociceptive-labeled neurons, and waited 3 days before applying CNO and conducting further behavioral experiments.
Article A nociceptive neuronal ensemble in the dorsomedial prefronta...
Shan Jiang et al. (2022) used the same strategy and also waited just 3 days after AITC-induced nociceptive labeling.
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On the other hand, Giuseppe Giannotti et al. (2019) used the [AAV8-cfos-ERT2-Cre-ERT2-PEST] and the [AAV2-DIO-hM4Di-mCherry] viruses and waited 28 days after fear conditioning before memory retrieval. Although they pointed to DeNardo's investigation, as far as I understand, such an amount of time is related to the assembly and incorporation of memory circuits, rather than sufficient DREADD expression.
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Li Ye et al. (2016) used the TRAP strategy for fluorescent labeling of active neurons, but only described the time schedule of their experiment (waiting for 3 weeks after TRAPing), without explaining the reasons.
Article Wiring and Molecular Features of Prefrontal Ensembles Repres...
As I lack knowledge about genes, vectors, and plasmids, I assume there is a fundamental reason that I cannot comprehend. Any insights or guidance in the right direction would be highly appreciated.
In my own view, you can check the expression at least 1-week after TRAPed. Once the mCherry is ovserved, the virus shall be expressed well done (Virus express sequentially, e.g. pAAV-hSyn-DIO-hM3D(Gq)-mCherry: AAV should be expressed first and the mCherry will be expressed at last. Only the Cre+ cells will be expressed the sequence after DIO).
Hope this will help.
Dear Jinming Zhang, Thank you for your reply.
I am just curious if any articles or studies explaining such a time frame exist or not. I thought there was a study about, let's say, changes in the portion of mCherry-expressing cells with time (e.g., 3 days, 7 days, 14 days after TM). If the portion of mCherry-positive neurons 7 days after TM is exactly the same as after 28 days, time consumption will be much less.
You can check the original article by Liqun Luo (Article Permanent Genetic Access to Transiently Active Neurons via T...
).Although they didn't describe the AAV strategy, we can still be inspired.
In the supplemental information, Figure S5 E-F show that the tdTomato expression induced by Fos reaches nearly 100% 72 hours after 4-OHT injection. So theroatically, I guess it shall be no difference between 7 days and 28 days ( In my exprience, AAV-carried fluorscenct protein may express faster than Cre-Loxp mice after TM or 4-OHT injection ).
Also Article Temporal evolution of cortical ensembles promoting remote me...
Thank you for your suggestion!
I have already seen these article. It is just that they use different strategy. In the article from Prof. Deisseroth where the [pAAV-cFos-ERT2-Cre-ERT2-PEST-no WPRE] was first introduced, they were waiting for 4 weeks because, as they mentioned in the methods section, "to allow the full expression of fluorophores". I'm just wondering if there are any experimental proofs that four weeks are necessary in the case of using that virus. It's probably better to ask them directly.
Article Wiring and Molecular Features of Prefrontal Ensembles Repres...
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