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Round Chemogenetics Icon Showing a Schematic of a Molecule Chemogenetics Guide


Chemogenetics refers to the engineering of protein receptors to respond to previously unrecognized small molecules. Chemogenetic tools are actuators for specific cellular pathways targeted to specific cell populations (most often neurons) that can be turned on or off by the application of a small molecule ligand. The ideal chemogenetic tools are unresponsive to native ligands and are engineered to respond to small molecules that do not affect endogenous signalling, therefore allowing precise control over the cell population they are targeted to.

Early Chemogenetic Receptors/RASSLs

The first chemogenetic receptors were based on G-protein coupled receptors, or GPCRs. The largest class of cell surface receptors, GPCRs are seven-pass transmembrane proteins that bind a specific ligand. Ligand binding then activates G-proteins to modulate downstream signalling. GPCRs have been shown to be involved in a wide variety of biological processes, including initiating signalling pathways in inflammation and neurotransmission. This, and the fact that GPCR ligands can have high potency, which is potentially important for dosing in animal studies, made them attractive targets for development into chemogenetic tools.

Need a refresher on G-protein coupled receptors? Read the GPCRs: How Do They Work and How Do We Study Them? blog!

Studies of GPCRs led to development of mutated receptors that while unresponsive to endogenous ligands, could be activated by synthetic ligands. Specifically, Receptors Activated Solely by Synthetic Ligands (RASSLs) based on κ-opiod receptors, were developed. These receptors were activated by the synthetic ligand spiradoline, however, spiradoline exhibited off-target effects in vivo and the receptors exhibited high levels of constitutive activity, making them less than ideal.


Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), like RASSLs, are based on engineered G-protein coupled receptors, but unlike RASSLs, show insensitivity to endogenous ligands, have low constitutive activity, and their activating ligands have few, if any off-target effects. They are the most widely employed type of chemogenetic receptors. There are several different types of DREADDs that can be broadly classified by the signalling protein that the receptors couple to:

Gq-DREADDs signal through the Gαq/11 G-protein and activate neuronal firing through stimulating phospholipase C, which releases intracellular calcium stores. There are currently three Gq DREADDs based on human muscarinic receptors: hM1Dq, hM3Dq, and hM5Dq, however, hM3Dq is the most widely employed.

Gi-DREADDs signal through the Gαi/o G-protein and inhibit neuronal signalling by inhibiting adenylate cyclase and downstream cAMP production. There are currently two Gi DREADDs based on human muscarinic receptors, hM2Di and hM4Di, and a Gi DREADD based on the human κ-opoid receptor, termed KORD (or KORDi).

Gs-DREADDs signal through the Gαs G-protein and activate neuronal signalling by increasing intracellular cAMP concentrations. There is currently one Gs-DREADD, rM3D, that was created by replacing the corresponding intracellular region of a turkey erythrocyte β-adrenergic receptor with a rat M3 muscarinic receptor. This DREADD was shown to have a small amount of constitutive activity, and is not widely used.

There is also a DREADD that couples to β-arrestin to activate noncanonical GPCR signalling independent of G proteins. This DREADD, termed Rq(R165L), is based on the human M3 muscarinic receptor and has not been used in vivo.

Schematic showing available DREADDs, what ligand they are activated by, and their effect on cellular signaling

Figure 1. DREADDs, their ligands, and signaling properties. Please see the text or table 1 for details.

DREADD Ligands

Muscarinic-receptor based DREADDs were engineered to respond to nM concentrations of clozapine N‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be pharmacologically inert in mice and rats. However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target effects. More recent studies have shown that CNO does not cross the blood-brain barrier in rats, and the utility of DREADDs in experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for alternative DREADD ligands.

Compound 21, Deschloroclozapine (DCZ), Perlapine, and Olanzapine have all been explored as alternative ligands for muscarinic-receptor based DREADDs. Compound 21 has similar potency as CNO, while DCZ is more potent than either CNO or Compound 21. Both Compound 21 and DCZ seem to have minimal off-target activity, and do not seem to have the same back-metabolism issues as CNO and are attractive alternatives for experiments. Perlapine has been previously used in human populations in Japan, making it an attractive option for translational studies, however, it has off-target effects. The atypical antipsychotic drug olanzapine has also been shown to be an agonist against hM4Di, and is especially attractive for use in translational studies because it is FDA and EMA approved.

One pitfall of a suite of muscarinic-based DREADDs that all respond to the same ligands was that bidirectional experiments (e.g. using both activating and inhibiting DREADDs in the same experimental setup) were not possible. To overcome this limitation, the inhibitory KORD DREADD was developed to respond to a different ligand, Salvinorin B (SALB). Scientists have shown that both KORD and hM3Dq can be used in the same organism to allow bidirectional control of neuronal activity.

Table 1. Descriptions of DREADDs and their activity in neurons

DREADD Receptor Effector Ligand Effect Outcome (in neurons) Reference
Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling Nakajima & Wess, 2012
hM3Dq hM1Dq hMD5q Human M3 muscarinic Gαq CNO* Increase Ca2+ Neuronal burst firing Armbruster et al, 2007
rM3D Rat M3 muscarinic & turkey β1-adrenergic Gαs CNO* Increase cAMP Neuronal burst firing Guettier et al, 2009
hM4Di hM2Di Human M4 muscarinic Gαi CNO* Decrease cAMP Neuronal inhibition Armbruster et al, 2007
KORD Human κ-opioid Gαi SALB Decrease cAMP Neuronal inhibition Vardy et al, 2015
*for a discussion of alternative DREADD ligands, please see the text

Browse Addgene's collection of DREADD plasmids!


Unlike DREADDs, which manipulate neuronal activity indirectly through GPCR signalling, another class of chemogenetic receptors that confer more direct control of neurons through manipulation of ion channels are Pharmacologically Selective Actuator Modules (PSAMs, pronounced SAMs). PSAMs are engineered α7 nicotinic acetylcholine receptor (nAChR) domains that respond to specific small molecules termed Pharmacologically Selective Effector Molecules (PSEMs). PSAM domains were first named for the mutations that allowed them to respond to their cognate PSEM. For example, PSAMQ79G,Q139G is activated by PSEM22S, while PSAML141F,Y115F is activated by PSEM89S. Later, in an effort to develop a PSAM that responded to a clinically approved drug, the PSAM domain was engineered to respond specifically to the anti-smoking drug varenicline. This PSAM carries mutations at L131G, Q139L, and Y217F and is termed PSAM4 (or PSAM4). Additional varenicline analogs, termed ultrapotent PSEMs (or uPSEMs), have also been developed as agonists for PSAM4. A given PSAM is coupled with an ion pore domain (IPD) to form ligand-gated ion channels (LGICs) that can either excite or inhibit neuronal signalling. PSAM-based LGICs can be classified by the ion pore domain they are coupled to:

PSAM-Gly LGICs pair a PSAM domain with a Glycine-receptor (GlyR) chloride-selective IPD. Binding of the cognate PSEM allows for the influx of Cl- ions, and inhibits neuronal activity.

PSAM-5HT3 LGICs pair a PSAM domain with a 5-HT3 serotonin receptor sodium- or potassium-selective IPD. Binding of the cognate PSEM allows for influx of Na+ and/or K+ ions, and activates neuronal activity.

Schematic showing PSAM4 based Ligand Gated Ion Channels, their effect, and outcome in neurons

Figure 2. PSAM4-based LGICs, their effect, and outcome in neurons. Please see the text or table 2 for details.

Table 2. Descriptions of PSAMs and their activity in neurons

PSAM Ion Pore Domain Ligand(s) Effect Outcome (in neurons) Reference
PSAM4 Gly Varenicline, uPSEM 792, uPSEM 817 Cl- influx Neuronal inhibition Magnus et al, 2019
PSAM4 5HT3 Varenicline, uPSEM 792, uPSEM 817 Na+, K+ influx Neuronal activation Magnus et al, 2019
PSAMQ79G,Q139G Gly PSEM22S Cl- influx Neuronal inhibition Magnus et al, 2011
PSAMQ79G,Q139G 5HT3 PSEM22S Na+, K+ influx Neuronal activation Magnus et al, 2011
PSAML141F,Y115F Gly PSEM89S Cl- influx Neuronal inhibition Magnus et al, 2011
PSAML141F,Y115F 5HT3 PSEM89S Na+, K+ influx Neuronal activation Magnus et al, 2011

Browse Addgene's collection of PSAM plasmids!

Planning your Chemogenetics Experiment

You're just starting your chemogenetics experiment planning. Here are some things to consider:

Chemogenetic activation or inhibition. Do you want to turn ON or turn OFF neurons in your experiment? Depending on your answer, you’d pick an activating or inhibitory chemogenetic receptor, respectively.

DREADDs or PSAMs. DREADDs are monomeric proteins, while PSAMs have more than one domain, both of which need to be expressed. However, neuronal control through PSAMs is direct, while neuronal control of DREADDs is indirect. DREADD ligands affect signalling for up to 8 hours after delivery, while PSEMs have an effect for only 0.5-1 hours after delivery.

Chemogenetic Ligand.The chemogenetic receptor chosen and the context of the experiment will determine the chemogenetic ligand used. For example, a study in rats using an hM4Di receptor might consider an alternative DREADD ligand such as DCZ versus CNO.

Delivery. Chemogenetic receptors are delivered in vivo through viral injection or use of genetically engineered mouse models. For viral injection, Adeno-associated virus (AAV) is a widely-used tool for achieving in vivo expression of chemogenetic receptors, and a wide variety of AAV-encoding chemogenetics plasmids are available. Additionally, genetically engineered animal models expressing hM3Dq, hM4Di, and rM3D DREADDs have been developed and are also commercially available.

Browse our collection of Ready-to-use AAV Preparations of Chemogenetic Plasmids!

Table 3. Promoters used in chemogenetics plasmids

Promoter Cell Specificity
hSyn1, CaMKIIa Neurons
CD68 Microglia
Dlx Interneurons
EF1a, CAG General expression

Localization. Depending on the experiment, an AAV-encoded chemogenetic plasmid may need to be targeted to specific tissues, cell types or even subcellular regions of a neuron. Cell-specific expression of AAV-delivered constructs can be controlled with cell-type specific promoters. Table 3 lists some common promoters found in chemogenetic receptor constructs and the cell types in which they drive expression.

Different AAV serotypes can be used to target AAV vectors to specific cells of interest. In addition, different AAV serotypes have varying degrees of viral spread within injected tissue, and this must be considered. For tissue specificity of AAV serotypes, see our AAV guide.

FLEx Vectors are also used to achieve cell-specific expression of AAV-encoded chemogenetic receptors. FLEx vectors block expression in the absence of Cre and are especially useful in AAV experiments where the virus can spread around the injection site and potentially express the chemogenetic receptor in unwanted cell types. Generating a FLEx switch to control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific Cre is provided.

Want to know more about FLEx vectors? Read our Plasmids 101: FLEx Vectors blog!

Download our chemogenetics poster!

screenshot of the chemogenetics poster, click to download


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