An Overview of Chemogenetics

Due to significant developments in neuroscience techniques, researchers are now able to selectively exploit neural systems in conscious animals, through emerging methods called chemogenetics and optogenetics. These methods help in exploring the neural circuitry underlying intricate behaviors in disease and health.

Chemogenetics and optogenetics show similarities in their approach of modifying neuronal activity; for example, in both techniques, ion channels or engineered receptors have to be introduced in particular brain regions, through plasmid expression or viral vector systems. In optogenetics, bacterial light-sensitive ion channels must be expressed and fiber optics also have to be subsequently used to inhibit or activate neuronal activity in vivo or in vitro (Boyden et al., 2005; Zhang et al., 2007).

Although this method offers superior temporal control of in vivo neuronal activity, it is known to be inherently invasive and needs cerebral implantation of fiber optics. Chemogenetics, on the other hand, does not need a chronic implant but maintains the potential to control neuronal activity. This is accomplished by administering ligands, selective for ion channels or engineered receptors, which are otherwise inert (Armbruster et al., 2007; Campbell & Marchant, 2018). Table 1 shows the main features of chemogenetics and optogenetics.

Table 1. Chemogenetics vs optogenetics

History and Development

RASSLs

Chemogenetics refers to the use of ion channels or genetically engineered receptors and the selective ligands activating those receptors to facilitate the manipulation of neuronal activity. In this context, G-protein coupled receptors (GPCRs) have been spearheading the development of chemogenetics, and the original paper defining GPCRs that react only to synthetic ligands was published in 1998.

These receptors — known as Receptors Activated Solely by a Synthetic Ligand (RASSLs) — were effectively applied in vivo, for instance, to remotely control cardiac activity. Despite this fact, the application of RASSLs in neuroscience has been limited by the endogenous activity of receptors in the absence of their particular ligand and the pharmacological activity of ligands in vivo (Coward et al., 1998; Sternson & Roth, 2014).

DREADDs

The recent years have seen the development of Designer Receptors Exclusively Activated by Designer Drugs (DREADDS). Mutated human muscarinic receptors stimulated only by inert ligands were the first DREADDS to be developed (Armbruster et al., 2007). Through several rounds of mutagenesis and screening against the biologically inert ligand Clozapine N-oxide (CNO), muscarinic receptors coupled to the G_αq intracellular signaling pathway were identified.

Receptors coupled to this pathway are capable of activating neuronal activity in response to CNO. Low concentrations of CNO activate all three G_αq-DREADDs — hM₁D_q, hM₃D_q, and hM₅D_q (Roth, 2016). Moreover, the same study revealed that hM₄D_i and hM₂D_i can inhibit neuronal activity via their coupling to G_αi intracellular signaling pathways. These inhibitory DREADDs also respond to CNO (Armbruster et al., 2007; Figure 1).

Figure 1. Mechanism of action of DREADD ligands. Binding of DREADD ligands to G_αq-DREADDs provokes neuronal firing, whereas binding to G_αi-DREADDs results in inhibition of neuronal activity. Clozapine N-oxide dihydrochloride and DREADD agonist 21 are non-selective muscarinic DREADD agonists and so can activate or inhibit neuronal activity, depending on the specific receptor being expressed. Salvinorin B is selective for the KORD receptor, which is coupled to G_αI signaling, consequently binding results in inhibition of neuronal activity. Image credit: Tocris Bioscience

The binding between G_αq-DREADDs and CNO causes the stimulation of phospholipase C (PLC), which catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP₂) to 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). Both DAG and IP₃ possess second messenger functions: the latter binds to its receptors to trigger the release of Ca²⁺ from intracellular stores, whereas the former stimulates various forms of protein kinase C (PKC).

The binding between G_αi-DREADDs and CNO causes the inhibition of adenylyl cyclase (AC), resulting in decreased intracellular cAMP levels. Since both EPAC and protein kinase A (PKA) are activated by cAMP, the CNO’s action at G_αi-DREADDs inhibits EPAC and PKA downstream signaling (see Figure 1).

CNO is a metabolite of clozapine, studies indicate that this is a bidirectional conversion and that CNO can undergo reverse metabolism to clozapine. When CNO is administered at the concentrations needed to activate DREADDs, clozapine is subsequently able to activate endogenous receptors (Gomez et al., 2017). Clozapine — an atypical antipsychotic — acts at an array of targets and leads to many different behavioral effects.

Rats, mice, humans, non-human primates, and guinea pigs all show the reversible metabolism of CNO to clozapine (Gomez et al., 2017; Manvich et al., 2018). As a result of potential reverse metabolism of CNO, structure-activity relationship studies have evolved to develop stable, alternative ligands.

The potent DREADD ligand — DREADD agonist 21 — was initially analyzed for activity against hM₃D_q. The approved drug perlapine was identified as a powerful hM₃D_q agonist in the same research. In Japan, this drug has been approved as a sedative and hypnotic (Chen et al., 2015). Both perlapine and DREADD agonist 21 have been subsequently shown to be potent agonists of hM₄D_i, hM₃D_q, and hM₁D_q with little to no off-target activity. Moreover, DREADD agonist 21 has been tested in vivo, in which it has been demonstrated to activate hM₃D_q-expressing neurons and inhibit the activity of hM₄D_i-expressing neurons (Thompson et al., 2018).

Subsequent to the development of muscarinic DREADDs, an inhibitory DREADD has been created from the κ-opioid receptor (KORD). Activation of this inhibitory DREADD is achieved through binding of the ligand Salvinorin B, resulting in the inhibition of neuronal activity through G_αi signaling. To allow bidirectional control of neuronal activity, KORD can be utilized alongside, activating DREADDs like hM₃D_q (Vardy et al., 2015).

Some common features in all DREADDs make them suitable for application in neuroscience experiments. Firstly, DREADDs do not exhibit any response to endogenous ligands because of genetic mutations within their ligand binding sites that eliminate binding, implying that any activity of the DREADD will be only because of the applications of the specific DREADD ligand. Secondly, in vitro or in vivo expression of DREADDs does not have any impact on baseline behaviors, neuronal function, or cellular activity, before the addition of the DREADD ligand (Sternson & Roth, 2014).

PSAMs/PSEMs

While DREADDs and RASSLs are based on GPCRs, modified ion channels named Pharmacologically Selective Actuator Modules (PSAMs), have also been used for modulating the activity of neurons. PSAMs are based on studies indicating that it is possible to transplant the extracellular ligand binding domain of the α7 nicotinic ACh receptor (nAChR) onto the ion pore domain of other ligand-gated ion channels. When the α7 nAChR ligand binding domain is spliced with the ion pore domain of the 5-HT₃ receptor, an ion channel with α7 nAChR pharmacology is produced but with 5-HT₃ cation conduction properties (Eiselé et al., 1993).

Likewise, splicing of the α7 nAChR ligand binding domain with the ion pore domain of the chloride selective glycine receptor (GlyR) produces an ACh responsive chloride channel (Grutter et al., 2005). Selective mutation of the α7 nAChR ligand binding domain consequently generates PSAM ion channels, which do not show any ACh binding, but are still selectively bound by compounds called Pharmacologically Selective Effector Molecules (PSEMs).

PSAMs or chimeric ion channels allowing regulation of anion or cation conductance have been produced through the combination of the mutated α7 nAChR ligand binding domain (harboring two or one mutations) with the ion pore domain of several varied ligand-gated ion channels. Such PSAM chimeras are named based on their mutations as well as linked ion pore domain — PSAM^L141F,Y115F-GlyR, PSAM^L141F-GlyR, PSAML^141F,Y115F-GABA_C, and PSAML^141F,Y115F-5-HT₃. Activation of neuronal activity is enabled by 5-HT₃-containing chimeras, whereas GABAC- and GlyR-containing chimeras are inhibitory (see Figure 2) (Magnus et al., 2011; Sternson & Roth, 2014).

Figure 2. Mechanism of action of PSEMs. Activating PSAMs are composed of a mutated α7 nAChR ligand binding domain spliced with the ion pore domain of a cation-selective channel, such as 5-HT₃. Binding of PSEMs to activating PSAMs results in an influx of cations and activation of neuronal activity. Inhibitory PSAMs are composed of a mutated α7 nAChR ligand binding domain spliced with the ion poredomain of an anion selective channel, such as GlyR. Binding of PSEMs to inhibitory PSAMs results in an influx of anions and inhibition of neuronal activity. Image credit: Tocris Bioscience

Scientific reviews for further reading

• Campbell & Marchant (2018) The use of chemogenetics in behavioral neuroscience: receptor variants, targeting approaches and caveats. Br J Pharmacol. 175, 994.
• Roth (2016) DREADDs for Neuroscientists. Neuron. 89, 683.
• Magnus et al. (2011) Chemical and genetic engineering of selective ligand-ion channel interactions. Science. 333, 1292.
• Sternson & Roth (2014) Chemogenetic tools to interrogate brain functions. Annu Rev Neurol. 37, 387.

References

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7. Coward et al. (1998) Controlling signaling with a specifically designed Gi-coupled receptor. Proc Natl Acad Sci USA. 95, 352.
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17. Thompson et al. (2018) DREADD agonist 21 (C21) is an effective agonist for muscarinic-based DREADDs in vitro and in vivo. ACS Pharmacol Transl Sci. Epub ahead of print.
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19. Varela et al. (2016) Tracking the time-dependent role of the hippocampus in memory recall using DREADDs. PLoS One. 11, e0154374.
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