g protein - coupled receptors ( gpcrs ) represent the largest family of membranous signaling molecules and are characterized structurally by a highly conserved seven - transmembrane domain motif ( allen and roth , 2011 ; meltzer and roth , 2013 ) . gpcrs recognize an incredible variety of ligands , including odorants , photons , neurotransmitters , lipids , hormones , peptides , and other small molecules , and regulate intracellular response to adapt to the ever - changing environment ( allen and roth , 2011 ) . upon agonist activation , gpcrs undergo prototypical structural re - arrangements facilitating the interaction with a family of canonical heterotrimeric g - proteins ( e.g. , gq , gs , gi , and g12/13 ) and g protein - independent signaling molecules ( allen and roth , 2011 ) . gpcr signaling modulates virtually every known physiological response ( allen and roth , 2011 ) to control gpcr signaling for neuropsychopharmacological therapeutics ,  selective  agonists or antagonists are typically developed ( besnard et al . , 2012 ; however , because most neuronal cells express multiple gpcrs and because most gpcrs are expressed in multiple tissues ( regard et al . , 2008 ) , it is essentially impossible to regulate a specific gpcr - signaling pathway in a selective cell population by known pharmacological approaches . additionally , the presence of endogenous receptor ligands and the lack of true specificity of pharmacological probes for gpcrs further confound the neuropsychopharmacological approach . to overcome these inherent problems associated with gpcr therapeutic approaches , the past two decades have seen a number of chemical - genetic approaches designed to control gpcr signaling in vivo ( see sternson and roth , 2014 , for recent review ) . although the  first generation chemogenetic tools were useful from a heuristic perspective , they were not widely adapted ( see sternson and roth , 2014 , for review ) . we adopted a novel strategy to discover useful chemogenetic tools utilizing directed molecular evolution in yeast . we were able to eventually evolve human muscarinic receptors to be activated by nm concentrations of the pharmacologically inert clozapine metabolite clozapine - n - oxide ( cno ) and to simultaneously be insensitive to the native ligand acetylcholine ( armbruster et al . , 2007 ) . importantly , the engineered receptors which we dubbed dreadds ( designer receptors exclusively activated by designer drugs ) had minimal constitutive activity in vitro ( armbruster et al . , 2007 ) and in vivo , even when expressed at high levels in neurons ( alexander et al . , 2009 ; zhu et al . , 2014 ) . since the initial description of dreadd technology ( armbruster et al . , 2007 ) , three dreadds have been mostly commonly used in the neurosciences : hm3dq , which is coupled to gq signaling and induces firing of neurons ( alexander et al . , 2009 ) ; hm4di , which is coupled to gi signaling and mediates neuronal ( armbruster et al . , 2007 ) and synaptic silencing ( stachniak et al . , 2014 ) ; and rm3ds , which is coupled to gs signaling and which modulates neuronal activity ( farrell et al . , 2013 ) . a schematic diagram for the application of dreadd technology is shown in figure 1 . the typical strategy for using dreadd technology to gain remote control over neuronal signaling is quite simple and is as follows ( see figure 1 for diagram of ways of expressing dreadds ) : schematic diagram for the application of dreadd technology in vivo . ( a ) two types of dreadd transgenic mouse models have been developed : cre - dependent and tet - dependent . dreadds can be expressed in genetically defined cell populations after crossing with cre- or tet - off driver mouse lines . ( b ) two types of dreadds viruses were developed : cre - dependent and cre - independent . in the cre - dependent system , virus containing double - floxed inverted dreadds are microinfused into the appropriate brain region in mice which express cell - type specific cre drivers . dreadds are flipped into the correct orientation and expressed after cre - mediated recombination to achieve very precise cell - type specific expression . express the dreadd in the cell - type of interest via either transgenic or viral technology for virally - mediated transduction wait 23 weeks for expression administer cno either via parenteral or oral administration within a few minutes of cno administration , the effect due to activation ( via hm3dq ) , inhibition ( via hm4di ) , or modulation ( via gsd ) of neuronal signaling occurs monitor behavior and/or neuronal firing in freely - moving animals the effects of cno will be negligible in animals which do not express dreadds and profound in animals expressing dreadds dreadds have been successfully demonstrated to control individual gpcr signaling in many in vivo studies from yeast , drosophila , mice , rats , and  perhaps in the near future  primates ( farrell and roth , 2012 ) and , as of august 2014 , more than 500 publications have cited dreadd technology . in the central nervous system ( cns ) , dreadds have been shown to control neuronal activities in a variety of neurons , including glutamatergic ( alexander et al . , 2009 ; zhu et al . , 2014 ) , serotonergic ( ray et al . , 2011 ) , dopaminergic ( dellanno et al . , 2014 ) , and gabaergic neurons ( kong et al . , 2012 ) , to name but a few . dreadds have also been used to control glial cell activity to modulate the autonomic nervous system ( agulhon et al . , 2013 ) . in periphery , dreadds have been used to control gpcr signaling in pancreatic beta - cells ( guettier et al . , 2009 ) , hepatocytes ( li et al . , 2013 ) , and breast cancer cells ( yagi et al . , 2011 ) . as these applications have been reviewed recently we will not comment on them further here ( urban and roth , in press ) . currently , a substantial collection of dreadd - based tools is available to control gpcr signaling in vivo ( see table 1 ) . three dreadd transgenic mouse lines created by the roth lab can now be obtained from jackson labs ( table 1 ) . cre - dependent hm3dq and hm4di transgenic mice are in the process of validation in the roth lab and will be made available soon to provide cell - type specific transgenic control of dreadd expression as outlined in figure 1 . specific expression of dreadds can also be achieved by viral delivery ( krashes et al . , 2011 ; urban and roth , in press ) . for many uses viral delivery is time - saving and yields a more precisely controlled dreadd expression . a variety of viral vectors  cre - dependent or -independent  are listed in table 1 and the majority of them can be obtained from unc viral core facility ( http://www.med.unc.edu/genetherapy/vectorcore/research-grade/ in - stock - aav - vectors / roth ) . current dreadd - based plasmids are available from addgene ( http://www.addgene.org/bryan_roth/ ) and as a service to dreadd users a blog is updated frequently ( http://chemogenetic.blogspot.com/ ) . summary of toolkit of dreadds technology we continue to develop new chemogenetic tools and soon will be describing those which afford bidirectional control of gpcr signaling in same - cell population via different synthetic ligands . in the meantime , new derivatives of dreadds have been developed by others which are likely to be quite useful . thus , for example , an arrestin - biased dreadd was generated ( nakajima and wess , 2012 ) and an axonal selective hm4d - neurexin variant was developed as a presynaptic silencer ( stachniak et al . , 2014 ) . the gpcr superfamily continues to be one of the major targets for drug discovery , although cns drug discovery remains slow and risky ( meltzer and roth , 2013 ) . indeed , less than 5% of cns drugs entering phase i clinical trials are eventually approved by the fda , and many gpcr - targeted drugs fail in phase ii and iii clinical trials due to lack of clinical efficiency ( bradley et al . , 2014 ) . this raises the questions as to whether current animal models really recapitulate the physiological / pathological condition seen in human disease and whether pharmacological intervention of disease - related gpcr targets can ultimately deliver the desired therapeutic response . for example , we can pre - evaluate whether pharmacological intervention of a given gpcr - signaling process by dreadd achieves the desired therapeutic response before starting a long and costly drug discovery process ( figure 2 ) . ( a ) introducing two mutations in transmenbrane iii and v of muscarinic receptors which create dreadds receptors that can precisely control the gq- , gi- , or gs - signaling pathways . ( b ) dreadds technology has been successfully applied in -cells , astrocytes , and a variety of neurons to control gpcr signaling in vivo . ( c ) dreadds technology has a great potential to be used in drug discovery , gene therapy , and tissue engineering . dreadds may also represent a novel approach for combining gene therapy , tissue engineering , and synthetic biology . ( 2014 ) demonstrated robust and bidirectional remote control of dreadd - expressing dopaminergic neurons , which induced from ipscs ( induced pluripotent stem cells ) , and were able to markedly enhance the beneficial effects in a mouse model of parkinson s disease . this represents the first demonstration of combining dreadd technology , gene therapy , and tissue engineering . theoretically this approach could be potentiated by crispr ( clustered regularly interspaced short palindromic repeats ) technology ( hsu et al . , thus , for instance , one could envision engineering ipscs by knocking in dreadds into the downstream regions of disease - related gpcr targets using crispr . dreadds could then be expressed at physiologically relevant levels in the correct cellular context . following transplantation , we could then remotely control engineered ipscs by administrating cno , a drug - like small molecule , to potentially cure human disease . , by engineering endogenous muscarinic receptors via crispr technology one could create mice in which endogenous muscarinic receptors are replaced by dreadds to achieve precise control over muscarinic receptor subtype signaling . this would allow researchers to deconstruct the roles of various muscarinic receptors in neuropsychiatric disease models . alternatively , crispr - based technology could be used to knock - in dreadds into essentially any locus in the genome to achieve cell - type specific control of signaling in virtually any tissue and organ . indeed , with the currently available tissue engineering , gene therapy , and synthetic biology tools we are limited only by our imagination in how we might adapt dreadd technology for interrogating and ultimately treating human neuropsychiatric diseases . all of the work involving dreadd technology in the roth lab summarized in this review has been supported by the nih and by narsad . mice expressing dreadd receptors have been licensed to takeda pharmaceuticals ; dr roth has received a grant from merck pharmaceuticals for enhancing dreadd technology . over the past 18 months dr roth has consulted for pfizer pharmaceuticals and ruiyi pharmaceuticals and has received honoraria from novartis pharmaceuticals .
recently , we created a family of engineered g protein - coupled receptors ( gpcrs ) called dreadd ( designer receptors exclusively activated by designer drugs ) which can precisely control three major gpcr signaling pathways ( gq , gi , and gs ) . dreadd technology has been successfully applied in a variety of in vivo studies to control gpcr signaling , and here we describe recent advances of dreadd technology and discuss its potential application in drug discovery , gene therapy , and tissue engineering .