A receptor activated solely by a synthetic ligand (RASSL) or designer receptor exclusively activated by designer drugs (DREADD), is a class of artificially engineered protein receptors used in the field of chemogenetics which are selectively activated by certain ligands. They are used in biomedical research, in particular in neuroscience to manipulate the activity of neurons. Originally differentiated by the approach used to engineer them, RASSLs and DREADDs are often used interchangeably now to represent an engineered receptor-ligand system. These systems typically utilize G protein-coupled receptors (GPCR) engineered to respond exclusively to synthetic ligands, like clozapine N-oxide (CNO), and not to endogenous ligands. Several types of these receptors exists, derived from muscarinic or κ-opioid receptors. Types of RASSLs / DREADDs One of the first DREADDs was based on the human M3 muscarinic receptor (hM3). Only two point mutations of hM3 were required to achieve a mutant receptor with nanomolar potency for CNO, insensitivity to acetylcholine and low constitutive activity and this DREADD receptor was named hM3Dq. M1 and M5 muscarinic receptors have been mutated to create DREADDs hM1Dq and hM5Dq respectively. The most commonly used inhibitory DREADD is hM4Di, derived from the M4 muscarinic receptor that couples with the Gi protein. Another Gi coupled human muscarinic receptor, M2, was also mutated to obtain the DREADD receptor hM2D. Another inhibitory Gi-DREADD is the kappa-opioid-receptor (KOR) DREADD (KORD) which is selectively activated by salvinorin B (SalB). Gs-coupled DREADDs have also been developed. These receptors are also known as GsD and are chimeric receptors containing intracellular regions of the turkey erythrocyte β-adrenergic receptor substituted into the rat M3 DREADD. RASSL / DREADD ligands A growing number of ligands that can be used to activate RASSLs / DREADDs are commercially available. CNO is the prototypical DREADD activator. CNO activates the excitatory Gq- coupled DREADDs: hM3Dq, hM1Dq and hM5Dq and also the inhibitory hM4Di and hM2Di Gi-coupled DREADDs. CNO also activates the Gs-coupled DREADD (GsD) and the β-arrestin preferring DREADD: rM3Darr (Rq(R165L). Recent findings suggest that systemically administered CNO does not readily cross the blood-brain-barrier in vivo and converts to clozapine which itself activates DREADDs. Clozapine is an atypical antipsychotic which has been indicated to show high DREADD affinity and potency. Subthreshold injections of clozapine itself can be utilised to induce preferential DREADD-mediated behaviors. Therefore, when using CNO, care must be taken in experimental design and proper controls should be incorporated. DREADD agonist 21, also known