hM4Di

hM4Di is a modified form of the human M4 muscarinic (hM4) receptor. It can be activated by the inert clozapine metabolite clozapine-N-oxide (CNO), engaging the Gi signaling pathway. Gi signaling in neurons opens potassium channels resulting in an influx of potassium ions that decreases the resting membrane potential of neurons and their capacity to depolarize. Thus, neurons expressing hM4Di treated with CNO are observed to have dramatically decreased firing rates¹.

Below you can find the plasmid maps provided from the Roth laboratory. Promoters are denoted in green, DREADDs are denoted in red and other proteins are denoted in brown. Some plasmids contain a full sequencing alignment. You can open the alignment panel through the sidebar on the right:

Mouse strain

• R26-LSL-Gi-DREADD (Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}; Jackson Laboratory #026219)
  • These R26-LSL-Gi-DREADD mice allow Cre recombinase-inducible expression of a CAG promoter-driven HA-hM4Di-pta-mCitrine from theGt(ROSA)26Sor locus.

AAV plasmids

CRE-dependent

Fused

• pAAV-EF1a-FLEX-HA-hM4D(Gi)-mCherry (Addgene #50461, map)
• pAAV-hSyn-FLEX-hM4D(Gi)-mCherry (map)
• pAAV-hSyn-FLEX-hM4D(Gi)-mCitrine-1BCT (JGE203, map)
• pAAV-hSyn-FLEX-hM4D(Gi)-mCitrine-2ACT (JGE204, map)

Unfused

• pAAV-hSyn-FLEX-HA-hM4D(Gi)-IRES-mCitrine (Addgene #50455, map)
• pAAV-hSyn-FLEX-HA-hM4D(Gi)-p2a-mCitrine-t2a-GIRK1 (map)

Recombinase-independent

Fused

• pAAV-CaMKIIa-hM4D(Gi)-mCherry (Addgene #50477, map)
• pAAV-GFAP-hM4D(Gi)-mCherry (Addgene #50479, map)
• pAAV-hSyn-HA-hM4D(Gi)-mCherry (Addgene #50475, map)
• pAAV-hSyn-hM4D(Gi)-mCherry (map)

Unfused

• pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine (Addgene #50467, map)
• pAAV-GFAP-HA-hM4D(Gi)-IRES-mCitrine (Addgene #50471, map)
• pAAV-hSyn-HA-hM4D(Gi)-IRES-mCitrine (map)

Lentiviral vectors

CRE-dependent

Fused

• pLenti6.3/V5-CMV-FLEX-HA-hM4D(Gi)-mCherry (map)

Unfused

• Not available

Recombinase-independent

Fused

• pLenti6.3/V5-CMV-HA-hM4D(Gi)-mCherry (map)

Unfused

• Not available

You can find pAAV plasmids without inserts for negative control here.

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References

1. Armbruster, Blaine N et al. "Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand." Proceedings of the National Academy of Sciences (2007).