DREADDs in rats

I occasionally get questions regarding approaches for using DREADDs in rats.  There are now a number of papers wherein cell-type specific expression of DREADDs in neurons was achieved using several platforms:


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CNO in drinking water

We frequently get requests regarding protocols for putting CNO in the drinking water for long-term studies. Here is our protocol: 1. Dissolve the CNO in a small volume of DMSO 2. Diluted the dissolved CNO in drinking water (e.g., 5mg/200ml) 3. Give mice with CNO drinking water  and protect from light using foil-wrapped bottles 4. The mice will receive 5mg/kg/day CNO (assume that mice weight 30g and consume 6ml water per day) 5. Prepare fresh daily. 6. A small amount of saccharine in the drinking water will mask the slightly bitter taste of CNO
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How best to dissolve CNO ...it depends on what it is soluble in (and that depends on the particular polymorph)

I recently received the following question: " My question is with regard to the use of CNO in drinking water (in mouse studies). Your recipe calls for the addition of a small amount of DMSO to dissolve CNO at a concentration of 5 mg in 200 mL. As we and others routinely dissolve CNO in 0.9% saline (no DMSO) at concentrations up to four times this amount, I am curious whether the addition of DMSO to CNO solutions for H2O drinking is critical, or whether the properties of CNO permit it's direct dissolution in H2O at concentrations from 5-20 mg per 200 mL. There appears to be no difficulty dissolving such concentrations in H2O in my hands, but I would prefer to hear your perspective. Is there a published reference for the solubility profile of CNO in H2O or 0.9% saline?" It basically depends upon the source of the CNO.  CNO via the NIH RAID program (which is what we use) is not readily soluble in H20 while commercial CNO is.  Although we've verified that both source...
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Considerations for immunofluorescence for various DREADD constructs

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I receive frequent queries regarding the appropriate way to visualize DREADDs.  Below I show the three basic DREADD constructs: HA-tagged mCherry fusion proteins HA-tagged in a vector with IRES-mCitrene In unfixed tissue the mCherry and mCitrene can be visualized using the parameters from the table below or by going here . NOTE: only the mCherry fusion protein fluorescence will show that the DREADD receptor is expressed.  mCitrene simply will show which cells were transfected or transduced. For visualizing HA we have a detailed recommended protocol   the principle of which is below. I also show various secondary antibodies which might be used along with excitation/emission characteristics Here's what you might consider if you have an mCherry fusion protein: Finally, what if you are using one of the HA-hM3Dq-IRES-mCitrene or HA-hM4Di-IRES-mCitrene constructs. ...
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