I'm new to DREADD, too, so I'm flagging this thread to see if there are interesting answers. I think that the problem with not using Cre mice will be that the vector will get into all the cells. You will get a blur with your mCherry reporter gene and CNO will inhibit everything in that area.

Why don't you want to use Cre?

Thanks for the response.

Yes, I know Cre is used to tag specific cell types, but in my case I just want a way to repeatedly inhibit all cells in the hippocampus without causing damage over time (e.g. from injecting into cannula many times). Ideally, we would use two different DREADDs with two different ligands which would allow us to independently inhibit dorsal vs. ventral hippocampus. Then, once we've (hopefully) established which region is more important regarding our behavioral task, we could focus on a specific cell type in a new group of animals.

Also, we have a group of animals currently trained on our task and just recently started thinking about DREADDs (thus, they are not transgenic).

So my hope with the original question is just to make sure the vector I have in mind is appropriate for this situation (i.e. inhibiting all cells in hippocampus).

Hi Ryan,

In mice, AAVs with the CaMKIIa promoter work quite well for expressing in forebrain excitatory/glutamatergic pyramidal neurons. Our lab has had good success with that approach in both dorsal and ventral hippocampus.

Hope this helps,

Ezequiel

If you want to inhibit two populations of neurons, you may want to use hM4Di DREADD for one population and PSAM-GlyR for the other. They use different ligands: CNO and PSEM895.

Shapiro MG, Frazier SJ, Lester HA. Unparalleled Control of Neural Activity Using Orthogonal Pharmacogenetics. ACS Chemical Neuroscience 2012; 3:619-629.

Thank you both for your advice.

Ezequiel - Do you know if it is common practice to specifically target pyramidal cells in the hippocampus when inhibiting hippocampal activity? Is there a specific reason you targeted pyramidal cells as opposed to simply inhibiting all neurons with an hSyn promoter? Just curious.

Juan - Thank you for this source. I have also heard of people using salvinorin B and KORD alongside hM3Di and CNO in order to control to different brain areas. Do you think there is any reason to choose GlyR over KORD?

The response of the GlyR is fast and straightforward because it is a Cl- channel that will hyperpolarize the neuron. The response to KORD, like that to hM4Di, is mediated through G proteins and K+ channels. Therefore, the responses of individual neurons depend on them expressing the right K+ channels and competition for the G proteins by other GPCRs.

I think what Ezequiel was pointing at is that if you inhibit all neurons, you will also inhibit the GABAergic interneurons, potentially inducing disinhibition. So, if you want to stimulate the hippocampus, it makes more sense to target only the excitatory neurons as he suggested.

I see, this makes sense now. Thank you for the explanation!

Hi Ryan,

it would be a much more straight forward approach to inhibit directly pyramidal neurons than having a GABA component, specially considering the high abundance of GABA neurons in the HPC (and all the different types). That is the reason why I think a CaMKIIa driven DREADD is a slightly better approach. This approach would also allow you to independently test if GABA mediated inhibition can contribute to your specific behavior/test; for that you could use mDlx (forebran GABA neuron) driven DREADDs (hM3Dq). We are also using this type of approach in our lab with very good results.

Ezequiel

This is interesting and relevant question. Are you able to succeed in doing DREADD in rats?

Hi Abdulghani,

there are a number of papers that have shown DREADDs working in rats; it should not be difficult to find them in Pubmed. In my field one of the authors using this approach is Gary Aston-Jones.

Hope this helps,

Ezequiel

Thanks Ezequiel, Where do you recommned to get the vector from?

In the US there are a number of places, Addgene would be a good starting point. Upenn or Stanford vector cores are also options although UPenn vector core has started already having its viruses available through Addgene.

I produce the AAVs for our own lab use as well as for University of Minnesota investigators but I have used both Addgene's and UPenn's in the past with good results.

Ezequiel

Hi Ryan,

I am also starting now with DREADDs in Long Evans Rats and in vHPC. I still haven t figure out the right non-Cre-dependent vector to use but I can keep you updated. Have you already decided ?

Hi Lorenzo,

Right now, I am thinking about using one or both of these two vectors:

https://www.addgene.org/50477/

https://www.addgene.org/65418/

However, Addgene only has the second one available as a plasmid. I've been searching around for someone who has this vector in stock, but have been unsuccessful. Several labs I've contacted can make the vector, but it is very expensive.

From what I've heard/read, the CaMKIIa promoter is the best choice for inhibitng hippocampal cells.

Thanks a lot Ryan,

My searching is taking me on the same direction. I asked to the

Byungkook Lab and I am waiting for an answer.

I ll let you know more as soon as I have news.

Hi Ryan Troha !

I was wandering if you ended up using the AAV8-CamKIIa-hM4D(Gi)-mCherry on your experiments. If you did, could you tell me which minimum expression period you used?

I will be using the same construct in dorsal hippocampus ( in rats) and I am trying to find an optimum expression period given the amount of virus injected.

Thanks

Hi Sofia,

We actually had some issues with our experiment and haven't used DREADDs yet. However, I believe most labs wait about 4 weeks after injecting the virus to allow for expression. Here's one source where they did this:

Varela, C., Weiss, S., Meyer, R., Halassa, M., Biedenkapp, J., Wilson, M. A., … Bendor, D. (2016). Tracking the Time-Dependent Role of the Hippocampus in Memory Recall Using DREADDs. PloS One, 11(5), e0154374.

I would also read this paper (below) if you haven't already about using low doses of Clozapine instead of CNO. Some colleagues of ours tried this in mice recently and it worked well for them.

Gomez, J. L., Bonaventura, J., Lesniak, W., Mathews, W. B., Sysa-Shah, P., Rodriguez, L. A., … Michaelides, M. (2017). Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science (New York, N.Y.), 357(6350), 503–507.

Hope this helps!