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Primer

Planning Your Next Chemogenetic Experiment

Mar 07, 2022

MaryAnn Labant
©: Anchor Therapeutics and Arkitek Studios

Chemogenetic experiments have been used to study a wide variety of behaviors, such as triggers for substance abuse, eating disorders, obesity, obesity-associated metabolic abnormalities, long term memory, hypertension, locomotor ability, as well as pain, startle, and fear responses. For instance, Horii-Hayashi and Nishi used chemogenetic activation of neurons in the hypothalamus of mouse models to uncover their role in risk assessment and burying behaviors toward a novel object. 1

Ideally, chemogenetic tools are engineered to respond to small molecules that do not affect endogenous signaling. These engineered receptors and biologically-inert ligands allow for the reversible remote control of cell populations and neural circuitry. Although temporal resolution is lower than with optogenetics, chemogenetics is relatively noninvasive and effective for functional mapping, cell-type-specific manipulations, and multiplexed control of neurons.2

Advances in chemogenetic receptors

GPCRs (G protein-coupled receptors) are involved in a wide variety of biological processes, including the initiation of signaling pathways in inflammation and neurotransmission.2 Their first use for chemogenetic studies was in 1991, introducing the concept of modifying endogenous receptors to alter their specificity and binding properties to permit activation at a chosen experimental time point.3

Studies using GPCRs led to development of Receptors Activated Solely by Synthetic Ligands (RASSLs), mutated receptors that could be activated by synthetic ligands, specifically spiradoline. However, spiradoline exhibited off-target effects in vivo and the receptors exhibited high levels of constitutive activity, making RASSLs less than ideal.4

Directed molecular evolution of GPCRs led to the development of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) for selective manipulation of cell activity through G protein signaling pathways. DREADs have low constitutive activity and their activating ligands exhibit few off-target effects. 

These muscarinic-based receptors are insensitive to the endogenous ligand (ACh), and show high affinity for clozapine and clozapine N-oxide (CNO). DREADDs have been developed that can either increase or decrease neuronal activity. The Gq DREADD increases neuronal firing by stimulating phospholipase C, releasing intracellular calcium stores. The less commonly used Gs DREADD stimulates cAMP production while Gi DREADD inhibits cAMP production.4

An experimental design limitation is that the same ligand activates both excitatory and inhibitory receptors rendering selective manipulation of neurons within the same model impossible. An inhibitory κ-opioid-based receptor DREADD (KORD) activated by the ligand salvinorin B (SALB) addresses this limitation.2

Although DREADDs are powerful for selectively manipulating a specific neuronal (or non-neuronal) subpopulation, studies indicate that ligands used for DREADDs, such as CNO or its parent compound clozapine, are not as selective as expected and sluggish kinetics, metabolic liabilities, and potential off-target effects of CNO represent areas for improvement.4,5 One such improvement is the new high-affinity and selective agonist deschloroclozapine (DCZ). 6

Another class of chemogenetic receptors known as Pharmacologically Selective Actuator Modules (PSAMs) confers more direct control of neurons through manipulation of ion channels. PSAMs are engineered α7 nicotinic acetylcholine receptor (nAChR) domains that respond to specific small molecules, Pharmacologically Selective Effector Molecules (PSEMs).4

 

Design considerations in a chemogenetic study

According to Addgene, there are several basic experimental design considerations. Foremost is the goal of the study—chemogenetic activation, inhibition, or a combination of both and the choice of receptor, DREADDs, or PSAMs

DREADDs are monomeric proteins and neuronal control is indirect. Neuronal control is direct with PSAMs but these receptors have more than one domain that need to be expressed. The experimental timeline is also critical. DREADD ligands affect signaling for up to eight hours after delivery, while PSEMs have an effect for only 0.5–1 hour. The chemogenetic receptor and experimental context will determine the ligand.4

Chemogenetic receptors are delivered in vivo through adeno-associated virus (AAV) injection or use of genetically-engineered mouse models. A wide variety of AAV-encoding chemogenetic plasmids are available.

Cell-type specific promoters can control cell-specific expression of AAV-delivered constructs. FLEX (FLip and Excise) vectors are also used to achieve cell-specific expression of AAV-encoded chemogenetic receptors. This widely adopted technique restricts expression of DREADD receptors to cells that express Cre.2

Most importantly, before any behavioral experiment the experimental conditions must be tested to determine the best ligand and optimal dose(s) in the system of interest. And, when possible, two different DREADD ligands should be tested to confirm that the observed behavioral effects are specifically DREADD mediated. Lastly, conventional pharmacological controls must be used. Groups of transgenic animals without expressing DREADDs (e.g., DREADD empty viral vectors) must be integrated to verify the selective effects of the ligand and chosen dose.4

References

  1. Horii-Hayashi N and Nishi M. “Protocol for behavioral tests using chemogenetically manipulated mice.” STAR Protocols. Volume 2, Issue 2. (2021): 100418. https://doi.org/10.1016/j.xpro.2021.100418
  2. Campbell EJ and Marchant NJ. “The use of chemogenetics in behavioural neuroscience: receptor variants, targeting approaches and caveats.” British Journal of Pharmacology. 175(2018): 994–1003.
  3. Chemogenetics Guide, Addgene website: www.addgene.org/guides/chemogenetics
  4. Goutaudier R, Coizet V, Carcenac C, and Carnicella S. “DREADDs: The Power of the Lock, the Weakness of the Key. Favoring the Pursuit of Specific Conditions Ratherthan Specific Ligands.” eNeuro. Sep-Oct; 6(5) (2019: ENEURO.0171-19.2019.  doi: 10.1523/ENEURO.0171-19.2019
  5. Gomez JL, Bonaventura J, Lesniak W, Mathews WB, Sysa-Shah P. et al. “Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.” Science. Aug 4;357(6350) (2017):503-507. doi: 10.1126/science.aan2475
  6. Nagai Y, Miyakawa, N, Takuwa, H, et al. “Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys.” Nat Neurosci 23 (2020): 1157–1167. https://doi.org/10.1038/s41593-020-0661-3
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