Skip to main content


What is Chemogenetics?

Chemogenetics is a powerful tool for activating or inhibiting cellular pathways within specific cell types, usually used in neuroscience to control neuronal or glial cell activity. Chemogenetics techniques use compounds that can selectively target either a genetically-modified GPCR (DREADD) or a ligand that acts at a chimeric ion channel. The mutant GPCRs are referred to as Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and their ligands are DREADD ligands. Chimeric ion channels are known as Pharmacologically Selective Actuator Modules (PSAMs) and their ligands are Pharmacologically Selective Effector Molecules (PSEMs).

The early chemogenetic receptors, receptors activated solely by synthetic ligands (RASSLs), were discovered by studying GPCRs. Mutated receptors were found that were unresponsive to endogenous ligands but could be activated by synthetic ligands. DREADDs are similar to RASSLs in that they are insensitive to endogenous ligands but they have low constitutive activity, compared to the high levels of constitutive activity of RASSLs. The activating ligands of DREADDS also have fewer off-target effects compared to RASSLs.

Chemogenetics is used in research to study a range of conditions including neurological and neurodegenerative disorders, pain, analgesia and epilepsy, and may be used alongside electrophysiology.

DREADDs and DREADD Ligands

Mechanism of action of DREADD ligands, like DREADD agonist 21, at DREADDs, like hM4Di

Figure 1. DREADD technology: mechanism of action and signaling pathways

DREADD Mechanism of Action

DREADDs were first developed from human muscarinic acetylcholine receptors (mAChRs). By introducing mutations at the orthosteric site, the affinity of the receptors for endogenous acetylcholine (ACh) is abolished, whilst maintaining responsiveness to small molecule DREADD ligands such as Clozapine-N-Oxide (CNO, Cat. No. 4936) or Deschloroclozapine (DCZ, Cat. No. 7193).

There are several types of DREADD, as defined by the G protein they are coupled to, see Table 1 below for a summary.

Table 1. DREADDs, their G-proteins, effect of activation and ligands.


G protein







Increase Ca2+ (excitatory)


CNO dihydrochloride

DREADD agonist 21 (Compound 21)

DREADD agonist 21 dihydrochloride




JHU 37152

JHU 37160




Decrease cAMP (inhibitory)



Decrease cAMP (inhibitory)

Salvinorin B



Arrestin translocation




Increase cAMP



DREADD Signaling Pathways

hM1Dq, hM3Dq and hM5Dq are coupled to the Gq signaling pathway; ligand binding activates phospholipase C, which catalyzes cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 and DAG both function as second messengers: IP3 induces Ca2+ release from intracellular stores by binding to intracellular receptors, while DAG activates multiple forms of protein kinase C (PKC).

Upon ligand binding, DREADDs coupled to the Gi signaling pathways cause inhibition of adenylyl cyclase (AC) - this leads to a decrease in intracellular cAMP levels. cAMP usually activates protein kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC), therefore ligand binding at Gi DREADDs inhibits PKA and EPAC downstream signaling (Figure 1). Gi-coupled DREADDs are also known as inhibitory DREADDs (iDREADDs) and include hM4Di and κ-opioid receptor DREADD (KORD).

KORD is an iDREADD developed from the κ-opioid receptor and is activated by the pharmacologically inert small molecule Salvinorin B (Cat. No. 5611). The simultaneous use of KORD and hM3Dq with their associated DREADD ligands enables researchers to have bidirectional control over neuronal activity, and allows simultaneous interrogation of κ-opioid and mAChR signaling.

GPCRs can also be coupled to β-arrestin-mediated signaling pathways, and function independently of G-proteins. β-arrestin interacts with and brings proteins into proximity, allowing downstream signaling through ERK. Rq(R165L) is an M3 muscarinic receptor with a point mutation that can selectively activate the β-arrestin pathway. However, the relatively high concentration of CNO required to activate Rq(R165L) receptors is likely to activate off-target receptors.

An additional DREADD derived from the human free fatty acid receptor 2 (FFA2) has been developed. This receptor bears mutations that alter the length of free fatty acid that can act on it; FFA2-DREADD responds to Sorbic acid (Cat. No. 7119), but not short chain free fatty acids. By using FFA-DREADD knockin mice, DREADD technology research is ongoing to investigate the role of FFA2 in the gut and gut-brain axis.

A newly discovered system uses Abscisic Acid (Cat. No. 6554), a phytohormone produced by plants and a range of animals, and found in mammalian tissue as a dietary component. Abscisic acid can induce interaction between ABI1 and PYL1 plant proteins. By fusing ABI1 to an hM3Dq receptor and PYL1 to β-arrestin2, abscisic acid can then be applied to induce membrane translocation of β-arrestin and resulting non-canonical signaling.


DREADD Ligand Evolution: CNO and Alternatives

The most commonly used DREADD ligand is CNO, which is a metabolite of Clozapine (Cat. No. 0444). Clozapine is an atypical antipsychotic that acts as an antagonist at dopamine and 5-HT2A/2C receptors. Research shows that CNO may undergo reverse metabolism to clozapine in vivo and that this may confound experimental results. An additional confounding factor is that CNO is a substrate for the P-glycoprotein (P-gp) multidrug transporter, which is both located on and helps to maintain the integrity of the blood brain barrier, and prevents the entry of drugs and removes them from the CNS.

The off-target effects of CNO and clozapine necessitated the development of alternative DREADD ligands. DREADD agonist 21 (Cat. No. 6422) is a potent hM3Dq and hM4Di DREADD agonist that displays excellent blood brain barrier penetration. Deschloroclozapine (DCZ, Cat. No. 7193) is a high affinity, highly potent activator of hM3Dq and hM4Di DREADDs, which displays low 'off-target' binding. DCZ is suited to in vivo use as it displays 100-fold greater affinity for hM3Dq and hM4Di DREADDs compared to CNO, and exhibits rapid (approximately 10 minutes after intraperitoneal injection) binding and activation of hM3Dq and hM4Di in mice and non-human primates.



The mechanism of action of PSEMs at PSAMs
Figure 2. Mechanism of action of PSEMs at PSAMs

PSAM and PSEM Mechanism of Action

GPCR-based tools such as DREADDS and DREADD ligands allow control of neuronal activity by inducing or inhibiting intracellular signaling pathways and altering the flux of ions through ion channels. PSAMs are chimeric ion channels that offer an alternative to GPCR-based tools and are a more direct way to manipulate ion flux and neuronal activity.

PSAMs were developed by transplanting the ligand binding domain of the α7 nicotinic acetylcholine receptor (nAChR) onto the ion pore domain of another ligand-gated ion channels (5-HT3 and glycine receptors). This produces a chimeric ion channel that has α7 nAChR ligand binding properties combined with the ion permeability properties of the ion pore domain used: 5-HT3 is an excitatory cation-selective pore; glycine is an inhibitory chloride-selective receptor.

Specific mutations are introduced to the α7 nAChR ligand binding domain to abolish binding of endogenous acetylcholine, which results in PSAMs that are only bound by inert and specific PSEM ligands. The PSAM ligand binding domain may have one or several mutations and PSAMs are named according to the mutations and the linked ion pore domain.

The effect that PSEM binding has on neuronal signaling depends on the receptor from which the ion pore domain of the PSAM originates. For example, PSEM 308 (Cat. No. 6425) binding to PSAML141F,Y115F-5-HT3 leads to neuronal activation via cation influx, while PSEM 308 binding to PSAML141F-GlyR inhibits neuronal activity through anion influx.


PSAM4 Chimeric Ion Channels and Ultrapotent PSEMs

Further research led to the development of ultrapotent PSEMs, known as uPSEMs, and potent PSAMs, called PSAM4. PSAM4-5HT3 and PSAM4-GlyR have mutations at L131G, Q138L and Y217F and are activated by very low nanomolar concentrations of Varenicline (Cat. No. 3754).

Compounds were later developed that were derived from varenicline and optimized to fit the PSAM4 ligand binding domain with higher specificity and potency. uPSEM 792 (Cat. No. 6865) and uPSEM 817 (Cat. No. 6866) are ultrapotent and selective agonists at PSAM4-5HT3 and PSAM4-GlyR. uPSEM 817 is also brain penetrant, which allows neuronal activity to be controlled non-invasively in vivo.


Expression of DREADDs and PSAMs

In order for DREADDs and PSAMs to be used, they must first be expressed in cells, either in cell culture or in vivo, and this is usually achieved through viral vector expression methods. Adeno-associated virus (AAV) is encoded with the mutant receptor and is then injected into the brain region of interest. It is possible to control the cell-type in which the receptor is expressed by using cell-specific genetic promoters. Examples of promoters include CaMKIIa, GFAP and CD68, which drive expression in neurons, glia and microglia respectively.

Chemogenetics Research Bulletin

This bulletin provides an introduction to the field of chemogenetics and chemogenetic methods to manipulate neuronal activity. It outlines the development of RASSLs, DREADDs and PSAMs, and the use of chemogenetic compounds. DREADD ligands and PSEMs available from Tocris are highlighted. Click below to download your copy today.