Hi Corey,

I'm not sure which behaviors you're looking at exactly, but I'm curious whether in the cohorts you use for the chronic experiments, you see the behavior in the first couple of CNO administrations? For example, maybe Days 1 through 3 the effect is robust but then it starts to fade? Your first bullet point might be getting at this, but it wasn't said what those experiments yielded.

Does it need to be DREADD-based inactivation? There are other methods you can use for cre-dependent inactivation or ablation like TeTox or Caspase, these might be easier for sustained inactivation than chronic hM4D stimulation.

Regarding the higher CNO doses, 5 mg/kg is already getting pretty high, even 1 mg/kg should be sufficient. For your initial cohorts, did you have non-hM4D controls that you gave CNO and did not observe your behavior of interest?

Best,

Justin

Hi Justin Siemian and thanks for your comments.

1) I have tested at 1 day, 3 days, and weekly up to 5 weeks, and cannot elicit the same behavioral changes when I administer CNO in drinking water (5mg/200mL), as I do with acute/semi-chronic injections (every 48-72 hours 30 min prior to testing). However, I haven't ruled out tolerance, as my acute CNO effects are prevented by prior chronic CNO exposure, as others have noted (Poyraz et al, J Neurosci, 2016).

2) I found DREADD attractive given that cells are preserved (but low functioning) and presynaptic neurotransmission is reduced, which accurately reflects my pathology of interest (excuse my vagueness). However, at this point I am open to a new approaches - would you mind sharing papers on those applications if you have them handy?

3) I agree that higher doses should be avoided. If I do go this route I would certainly use appropriate controls (i.e. all groups receive CNO). I did perform extensive tests to verify a lack of CNO effects in non-hM4D controls in a separate cohort.

Best,

Corey

Hi Corey,

I'm guessing you have already addressed this in your set-up - but CNO dihydrochloride is not stable long-term in solution at room temperature (see https://www.hellobio.com/stability-of-water-soluble-dreadd-ligands-in-solution-a-technical-review/).

FYI - we have also released some novel alternatives to CNO eg JHU 37160 ( https://www.hellobio.com/jhu37160-dihydrochloride.html), which has been used in vivo, and is water-soluble, which may be of interest.

Also - in terms of controls - there is a water-soluble clozapine now available too that might be of use: https://www.hellobio.com/clozapine-dihydrochloride.html

Regards

Sam