Dear Abdelrahman Mohsen Alkahwaji,

As others have already indicated, the timing between your CNO injection and  behavior experiment (10min post-injection) is likely too soon to have a behavioral effect. We've shown that 10mg/kg dose of CNO doesn't peak in cerebrospinal fluid until 90min after a subcutaneous injection in monkeys (http://pubs.acs.org.proxy.library.emory.edu/doi/abs/10.1021/acschemneuro.7b00079 ). Our study also demonstrated that CNO is metabolized into clozapine at levels that would activate the DREADD receptor. Last, but not least. Dr. Michael Michaelides recently published an article in Science demonstrating CNO is only activating the DREADD receptor through this conversion to clozapine (http://science.sciencemag.org.proxy.library.emory.edu/content/357/6350/503.long). Therefore, it's possible that not only is the timing between injection and behavioral test too early, but also that you may not have enough CNO being converted into clozapine to active your DREADD receptor.  I suggest first increasing your CNO dose and testing at 90 min post-injection.  I also suggest that you try low dose (0.1mg/kg) clozapine to activate the DREADD instead of CNO, as Dr. Michaelides demonstrates in his paper. However, the timing between clozapine injection and behavior will be much shorter (5-10min post-injection) because clozapine enters the brain more freely as compared to CNO.

If you have any further questions or need clarification on anything I said please let me know.   

Sincerely,

Jessica

I would recommend giving the drug 30 min before testing and 1-2 mg/kg doses. Perhaps these PFC areas are not directly implicatted in these behaviours and the tasks needs to be manipulated to show effects

I routinely use 0.3 mg/kg CNO i.p., 30 min prior to the experiment in both mice and rats. There are specific issues with getting CNO in solution, so keep this in mind. I tend to dilute right before injection, as it can often come out of solution, especially in more concentrated formulations in rats.

Be careful going above 1 mg/kg, as CNO can be behaviorally active at higher doses, 1 mg/kg is probably safe, but at least in rats 3 mg/kg can start to produce effects. I've seen some labs routinely use 5 and 10 mg/kg, this is really high and likely engaging non-specific mechanisms. There has been a recent paper showing that CNO doesn't cross the BBB, but is rather converted to clozapine, which can then cross and act on the DREADD, but at higher doses on native receptors. So it's important to use a behaviorally inactive dose (i.e. have a group with a control virus or at least CNO treated with no DREADD virus). 

Keep in mind that fluorescence doesn't necessarily equal adequate DREADD expression. Which virus did you use, how much did you inject?

Dear Abdelrahman Mohsen Alkahwaji,

As others have already indicated, the timing between your CNO injection and  behavior experiment (10min post-injection) is likely too soon to have a behavioral effect. We've shown that 10mg/kg dose of CNO doesn't peak in cerebrospinal fluid until 90min after a subcutaneous injection in monkeys (http://pubs.acs.org.proxy.library.emory.edu/doi/abs/10.1021/acschemneuro.7b00079 ). Our study also demonstrated that CNO is metabolized into clozapine at levels that would activate the DREADD receptor. Last, but not least. Dr. Michael Michaelides recently published an article in Science demonstrating CNO is only activating the DREADD receptor through this conversion to clozapine (http://science.sciencemag.org.proxy.library.emory.edu/content/357/6350/503.long). Therefore, it's possible that not only is the timing between injection and behavioral test too early, but also that you may not have enough CNO being converted into clozapine to active your DREADD receptor.  I suggest first increasing your CNO dose and testing at 90 min post-injection.  I also suggest that you try low dose (0.1mg/kg) clozapine to activate the DREADD instead of CNO, as Dr. Michaelides demonstrates in his paper. However, the timing between clozapine injection and behavior will be much shorter (5-10min post-injection) because clozapine enters the brain more freely as compared to CNO.

If you have any further questions or need clarification on anything I said please let me know.   

Sincerely,

Jessica