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Reference Icon Redbook Chemogenetics Guide


Chemogenetic receptors are powerful tools that can used to control and investigate cell signaling. While used in a variety of cell types, chemogenetic receptors are widely used in neuroscience. When introduced to the brain, these receptors can be used to investigate the relationship between behavior and particular neurons in the brain. Similar to optogenetics, wherein light is used to control neurons, chemogenetic receptors are activated by small molecules and in response, activate or inhibit neuronal firing. These receptors are typically unresponsive to native ligands and are engineered to respond to particular small molecules that are otherwise physiologically inert, and thus, enable specific control of neuronal activity with limited off-target effects. While popular in neuroscience, chemogenetic receptors have also been expressed in cardiac cells (Redfern et al. 1999), taste buds (Mueller et al. 2005, Zhao et al. 2003), pancreatic β-cells (Guettier et al. 2009), breast cancer cells (Yagi et al. 2011), and induced pluripotent stem (iPS) cells (Dell’Anno et al. 2014).

DREADD schematic

Schematic of various chemogenetic receptors and their signaling properties. Five types of chemogenetic receptors (Rq, hM3Dq, GsD, hM4Di, and KORD) have been genetically engineered from muscarinic or opioid receptors (as indicated by corresponding colors in the legend). Each receptor is specifically activated by its ligand (CNO, clozapine-n-oxide or SALB, salvinorin B) to signal to downstream effectors (Arrestin-2/Arrestin-3 or G-protein α subunits Gαq, Gαs, or Gαi). Activation of these effectors then leads to unique physiological outputs, as listed. Some receptors signal canonically through G-proteins (yellow box), while others have been engineered to signal through non-canonical pathways (green box).

G Protein-Coupled Receptors

To engineer chemogenetic receptors, researchers took advantage of G protein-coupled receptors (GPCRs), which are the largest group of membrane receptors in eurkaryotes, and the largest class of signal-transducing molecules in the brain. GPCRs are cell surface receptors that can intercept a variety of extracellular signals, including light, peptides, sugars, and lipids, and relay signaling to an intracellular G protein. The intracellular G proteins that associate with GPCRs comprised three subunits: the alpha, beta, and gamma subunits. In the resting state, the heterotrimeric G protein is bound to the GPCR and, in particular, the alpha subunit is in its inactive, GDP-bound state. Once a signal is received by a GPCR, it undergoes a conformational shift that activates the G protein, causing the exchange of GDP with GTP. The trimeric G protein now dissociates into two parts: the active, GTP-bound alpha subunit and the beta-gamma dimer complex, both of which which can then diffuse laterally (remaining bound to the plasma membrane) and signal to other membrane proteins.

Activated G proteins can signal to a variety of other proteins, and can activate production of second messengers. Each of the G protein subunits has different versions that have different binding partners, and thus, functions. There have been 5 beta-subunits, 11 gamma-subunits, and 20 alpha-subunits identified in mammals. Some of these G proteins activate their targets, while others can have inhibitory effects, and the combination of various G protein subunits to compose a G protein produces a diverse repetoire of G proteins and GPCR signaling within an organism. For example, the alpha subunit αs (Gαs) activates adenylate cyclase, causing production of the common second messenger cAMP. In neurons, cAMP elevation activates neuronal firing, while in smooth muscle, cAMP elevation causes muscle relaxation. Alternatively, the alpha subunit αi (Gαi) inhibits adenylate cyclase, and as a result can have an opposing effect (neuronal inhibition and smooth muscle contraction, respectively). Different alpha subunits can also have similar phenotypic outcomes. For example, the alpha subunit αq (Gαq) also causes smooth muscle contraction, but does so through the activation of phospholipase C. Thus, G proteins can activate a wide array of signaling pathways and lead to a variety of cellular responses.

As their name suggests, GPCRs couple to G proteins, and thus the activation of G protein is dependent on the activation of the GPCR. The activity of a GPCR can depend both on the ligand and on the downstream G protein that it couples to. GPCRs typically have a preference for one G protein subtype, but are capable of coupling to multiple subtypes. For example, the human muscarinic receptor M1 predominantly activates Gαq, but has also been shown to couple to Gαi and Gαs pathways. The human muscarinic receptor M1, however, has only been shown to couple with Gαq. In addition, different ligands can cause a GPCR to couple to different G proteins. Overall, the output of a GCPR depends on the ligand, the composition of the G protein to which the receptor couples, and the cell type in which the signaling occurs.

Chemogenetic Receptor Technologies

One major class of chemogenetic receptors is based on mutated GPCRs (Strader et al. 1991). These mutants were first created using wild-type β2-adrenergic receptor GPCRs that were engineered by site-directed mutagenesis to bind nonnatural ligands at high potency (e.g., at micromolar concentrations), while being unresponsive to native ligands (Small et al. 2001). Since nonnatural ligands exhibited off target effects and the engineered GPCRs exhibited high levels of constitutive activity (Sternson and Roth, 2014), the next phase of modified GPCRs were instead engineered to respond to specific, pharmacologically-inert, drug-like small molecules (Armbruster & Roth 2005, Alexander et al. 2009, Armbruster et al. 2007), a technology termed DREADD (designer receptor exclusively activated by designer drug). DREADDs, which were engineered from members of the human muscarinic receptor family, have the advantages of (1) nanomolecular potency for activation by the small molecule ligand clozapine-N-oxide (CNO), (2) relative insensitivity to the endogenous ligand acetylcholine, and (3) no detectable constitutive activity.

Table 1. Descriptions of DREADDs and their activity in neurons

DREADD Original receptor Effector Ligand Effect Outcome (in neurons) Reference
hM3Dq Human M3 muscarinic Gαq CNO Increase Ca2+ Neuronal burst firing Armbruster et al. 2007
hM4Di Human M4 muscarinic Gαi CNO Decrease cAMP β/γ-GIRK activation Neuronal inhibition Armbruster et al. 2007
GsD Rat M3 muscarinic & turkey β1-adrenergic Gαs CNO Increase cAMP Neuronal burst firing Guettier et al. 2009
Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO Increase Arrestin translocation Arrestin signalling Nakajima & Wess 2012
KORD Human κ-opioid Gαi SALB Decrease cAMP β/γ-GIRK activation Neuronal inhibition Vardy et al. 2015

The M3 muscarinic receptor couples to Gαq, and the DREADD is thus termed hM3Dq to indicate its selectivity for Gαq-mediated signaling pathways, which can activate neuronal burst firing as a result of increasing intracellular calcium levels. DREADDs based on the M1 and M5 muscarinic receptors also couple to Gα (and are termed hM1Dq and hM5Dq, respectively), whereas M2- and M4-DREADDs couple to Gαi G proteins (and are termed hM2Di and hM4Di, respectively). Since these muscarinic receptors, and therefore these DREADDs (Table 1), couple to different G proteins, they exhibit different physiological effects including neuronal activation (burst firing), neuronal inhibition, or neuronal modulation. In addition to canonical G-protein activation, GPCRs also activate β-arrestin-mediated signaling pathways (Luttrell et al. 1999). This non-canonical

Table 2. Serotype-dependent spreading profiles of AAV-mediated GFP expression in the marmoset cerebral cortex
(Adapted from Watakabe et al. 2015)

Serotype Spread for injection site (mm) (mean1 ± SD)
1 1.2 ± 0.2
2 0.7 ± 0.2*
5 1.3 ± 0.3
8 No statistical difference observed between AAV1, 5, 8, 9
9 1.2 ± 0.4

1Mean of GFP spread using CMV, CaMKII, and SynI promoter-driven GFP expression
*significantly different from AAV1/5/9 by ANOVA (p<0.001) and from AAV8 by Ryan’s method (p<0.05)

signaling pathway can be controlled by a mutated M3-muscarinic receptor, termed Rq(R165L), which selectively activates arrestin signaling without perturbing G-protein mediated pathways (Nakajima & Wess 2012). A DREADD technology based on the kappa-opioid receptor (KOR-DREADD), termed KORD, is an inhibitory receptor activated by the inactive drug-like metabolite salvinorin B (SALB) (Vardy et al. 2015). Because the KORD ligand is distinct from that of the other G-protein based DREADDs, multiplexed chemogenetic interrogation can be achieved by dual expression of the inhibitory KORD and the excitatory hM3Dq DREADD within a cell (Vardy et al. 2015). DREADDs commonly used in neuroscience are described in Table 1.

Another major class of chemogenetic receptors are also based on the KOR that have been engineered to be unresponsive to endogenous ligands, but could be activated by the KOR agonist spiradoline (Coward et al. 1998). This class of chemogenetic receptor, termed RASSL (receptor activated solely by synthetic ligand), is limited by the pharmacological activities of their ligands (e.g., spiradoline), and the fact that some RASSLs exhibit high levels of constitutive activity (Hsiao et al. 2008, 2011; Sweger et al. 2007). Ligand-gated ion channels (PSAMs and PSEMs) represent another class of tools that can be used to interrogate neuronal function, but have limited application outside of excitable cells (Magnus et al. 2014).

Controlling Chemogenetic Receptor Localization in Experiments

Chemogenetic receptors represent a powerful tool for potent and specific control of cell signaling. However, they must be targeted to specific cell types in order to establish the roles of these cells in particular phenotypes. Adeno-associated virus (AAV) is a widely-used tool for achieving in vivo expression of chemogenetic receptors, and AAV-mediated receptor expression can be controlled with AAV serotype and the identity of the transgene promoter.

AAV encoding specific chemogenetic receptors can be injected into specific brain regions, and can thus be used for in vivo neuronal expression in mice, rats, and primates. The serotype of the AAV used for in vivo transduction can impact and enhance the delivery of constructs to particular cell populations. In addition, when performing in vivo injections of AAV, serotype can affect the diffusion of the AAV from the injection site (Table 2). These data suggest that AAV2 may exhibit reduced spreading from the injection site relative to AAV1, 5, 8, and 9, which may be important for targeting DREADD delivery or expression. For tissue specificity of AAV serotypes, see our AAV guide.

Cell-specific expression of AAV-delivered constructs can be further controlled with cell-type specific promoters that drive receptor expression. Table 3 lists some common promoters found in chemogenetic receptor constructs and the cell types in which they drive expression.

Table 3. Description of promoters commonly used in chemogenetic plasmids

Promoter Description Cell Specificity
hSyn1 Fragment of the human synapsin 1 gene promoter Neurons
CaMKIIa Ca2+/calmodulin-dependent protein kinase II promoter Neurons
GFAP Glial fibrillary acidic protein gene promoter Glia
CD68 Human CD68 promoter Microglia
EF1a Strong mammalian expression from human elongation factor 1 alpha promoter General expression
CAG Strong hybrid mammalian promoter General expression

Finally, cell-specific expression of AAV-encoded receptors can be achieved by injecting vectors with loxP-type recombination site-flanked receptor sequences in mice with cell type-specific Cre recombinase expression (Urban and Roth 2015). These flip-excision (FLEX)-switch or double-inverted open reading frame (DIO) constructs essentially block receptor expression in the absence of Cre and can be used to achieve cell-specific expression across a range of cell types (Schnütgen et al. 2003, Atasoy et al. 2008, Kuhlman et al. 2008). For more information on FLEx vectors, see our FLEx Vectors blog post.



Alexander GM, Rogan SC, Abbas AI, Armbruster BN, Pei Y, et al. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63:27–39. PMID: 19607790

Armbruster B, Roth B. 2005. Mining the recepterome. J. Biol. Chem. 280:5129-32. PMID: 15590622

Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL. 2007. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. USA 104:5163–68. PMID: 17360345

Atasoy D, Aponte Y, Su HH, Sternson SM. 2008. A FLEX switch targets channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28:7025–30. PMID: 18614669

Coward P, Wada HG, Falk MS, Chan SD, Meng F, et al. 1998. Controlling signaling with a specifically designed Gi-coupled receptor. Proc. Natl. Acad. Sci. USA 95:352–57. PMID: 9419379

Dell’Anno MT, Caiazzo M, Leo D, Dvoretskova E, Medrihan L, et al. 2014. Remote control of induced dopaminergic neurons in parkinsonian rats. J. Clin. Invest. 124:3215–29. PMID: 24937431

Guettier JM, Gautam D, Scarselli M, de Azua IR, Li JH, et al. 2009. A chemical-genetic approach to study G protein regulation of β cell function in vivo. Proc. Natl. Acad. Sci. USA 106:19197–202. PMID: 19858481

Hsiao EC, Boudignon BM, Chang WC, Bencsik M, Peng J, et al. 2008. Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass. Proc. Natl. Acad. Sci. USA 105:1209–14. PMID: 18212126

Hsiao EC, Nguyen TD, Ng JK, Scott MJ, Chang WC, Zahed H, Conklin BR. 2011. Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice. Stem Cell Res Ther. 2:11. PMID: 21375737

Kuhlman SJ, Huang ZJ. 2008. High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression. PLoS ONE 3:e2005. PMID: 18414675

Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, et al. 1999. β-arrestin-dependent formation of β2 adrenergic receptor-Src protein kinase complexes. Science 283:655–61. PMID: 9924018

Magnus CJ, Lee PH, Atasoy D, Su HH, Looger LL, and Sternson SM, 2011. Chemical and genetic engineering of selective ion channel-ligand interactions. Science 333:1292–96. PMID: 21885782

Mueller KL, Hoon MA, Erlenbach I, Chandrashekar J, Zuker CS, Ryba NJ. 2005. The receptors and coding logic for bitter taste. Nature 434:225–29. PMID: 15759003

Nakajima K, Wess J. 2012. Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor. Mol. Pharmacol. 82:575–82. PMID: 22821234

Redfern CH, Coward P, Degtyarev MY, Lee EK, Kwa AT, et al. 1999. Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nat. Biotechnol. 17:165–9. PMID: 10052353

Schnütgen F, Doerflinger N, Calléja C, Wendling O, Chambon P, Ghyselinck NB. 2003. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 21:562–65. PMID: 12665802

Small KM, Brown KM, Forbes SL, Liggett SB. 2001. Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy. J Biol Chem. 276:1596-601. PMID: 11402033

Sternson SM, Roth BL. 2014. Chemogenetic tools to interrogate brain functions. Annu. Rev. Neurosci. 37:378–407. PMID: 25002280

Strader CD, Gaffney T, Sugg EE, Candelore MR, Keys R, et al. 1991. Allele-specific activation of genetically engineered receptors. J. Biol. Chem. 266:5–8. PMID: 1670767

Sweger EJ, Casper KB, Scearce-Levie K, Conklin BR, McCarthy KD. 2007. Development of hydrocephalus in mice expressing the Gi-coupled GPCR Ro1 RASSL receptor in astrocytes. J. Neurosci. 27:2309–17. PMID: 17329428

Urban DJ and Roth BL. 2015. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic Tools with Therapeutic Utility. Annu. Rev. Pharmacool. Toxicol. 55:399–417. PMID: 25292433

Vardy E, Robinson JE, Li C, Olsen RHJ, DiBerto JF, et al. 2015. A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior. Neuron. 86:936–46. PMID: 25937170

Watakabe A, Ohtsuka M, Kinoshita M, Tajaki M, Isa K, et al. 2015. Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex. Neurosci. Res. 93:144–57. PMID: 25240284

Yagi H, Tan W, Dillenburg-Pilla P, Armando S, Amornphimoltham P, et al. 2011. A synthetic biology approach reveals a CXCR4-G13-Rho signaling axis driving transendothelial migration of metastatic breast cancer cells. Sci. Signal. 4:ra60. PMID: 21934106

Zhao GQ, Zhang Y, Hoon MA, Chandrashekar J, Erlenbach I, et al. 2003. The receptors for mammalian sweet and umami taste. Cell 115:255–66. PMID: 14636554