The use of designer receptors exclusively activated by designer drugs (DREADDs) has led to significant advances in our understanding of the neural circuits that govern behavior. By allowing selective control over cellular activity and signaling, DREADDs have become an integral tool for defining the pathways and cellular phenotypes that regulate sleep, pain, motor activity, goal-directed behaviors, and a variety of other processes. In this review, we provide a brief overview of DREADDs and discuss notable discoveries in the neurosciences with an emphasis on circuit mechanisms. We then highlight methodological approaches to achieve pathway specific activation of DREADDs. Finally, we discuss spatial and temporal constraints of DREADDs signaling and how these features can be incorporated into experimental designs to precisely dissect circuits of interest.
AbelaARJiXDLiZLeADFletcherPJ. 2020. Clozapine reliably increases the motivation for food: parsing the role of the 5-HT2c and H1 receptors. Psychopharmacology (Berl)237:957–66.
2.
AghajanianGKBunneyBS. 1977. Dopamine “autoreceptors”: pharmacological characterization by microiontophoretic single cell recording studies. Naunyn Schmiedebergs Arch Pharmacol297:1–7.
3.
AlexanderGMRoganSCAbbasAIArmbrusterBNPeiYAllenJA, and others. 2009. Remote control of neuronal activity in transgenic mice expressing evolved G protein–coupled receptors. Neuron63:27–39.
4.
AndreoliMMarketkarTDimitrovE. 2017. Contribution of amygdala CRF neurons to chronic pain. Exp Neurol298:1–12.
5.
ArmbrusterBNLiXPauschMHHerlitzeSRothBL. 2007. Evolving the lock to fit the key to create a family of G protein–coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A104:5163.
6.
AtasoyDBetleyJNSuHHSternsonSM. 2012. Deconstruction of a neural circuit for hunger. Nature488:172–7.
7.
BaimelCLauBKQiaoMBorglandSL. 2017. Projection-target-defined effects of orexin and dynorphin on VTA dopamine neurons. Cell Rep18:1346–55.
8.
BecksteadMJGrandyDKWickmanKWilliamsJT. 2004. Vesicular dopamine release elicits an inhibitory postsynaptic current in midbrain dopamine neurons. Neuron42:939–46.
9.
Benoit-MarandMBorrelliEGononF. 2001. Inhibition of dopamine release via presynaptic D2 receptors: time course and functional characteristics in vivo. J Neurosci21:9134–41.
10.
BockaertJPinJP. 1999. Molecular tinkering of G protein–coupled receptors: an evolutionary success. EMBO J18:1723–9.
11.
BonaventuraJEldridgeMAGHuFGomezJLSanchez-SotoMAbramyanAM, and others. 2019. High-potency ligands for DREADD imaging and activation in rodents and monkeys. Nat Commun10:4627.
12.
BorglandSLTahaSASartiFFieldsHLBonciA. 2006. Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine. Neuron49:589–601.
13.
BrodnikZDXuWBatraALewandowskiSIRuizCMMortensenOV, and others. 2020. Chemogenetic manipulation of dopamine neurons dictates cocaine potency at distal dopamine transporters. J Neurosci40:8767–79.
14.
BrunelloNMasottoCSteardoLMarksteinRRacagniG. 1995. New insights into the biology of schizophrenia through the mechanism of action of clozapine. Neuropsychopharmacology13:177–213.
15.
CalebiroDKoszegiZ. 2019. The subcellular dynamics of GPCR signaling. Mol Cell Endocrinol483:24–30.
16.
CaoBJRodgersRJ. 1997. Dopamine D4 receptor and anxiety: behavioural profiles of clozapine, L-745,870 and L-741,742 in the mouse plus-maze. Eur J Pharmacol335:117–25.
17.
CassWAGerhardtGA. 1994. Direct in vivo evidence that D2 dopamine receptors can modulate dopamine uptake. Neurosci Lett176:259–63.
18.
ChenLYinDWangTXGuoWDongHXuQ, and others. 2016. Basal forebrain cholinergic neurons primarily contribute to inhibition of electroencephalogram delta activity, rather than inducing behavioral wakefulness in mice. Neuropsychopharmacology41:2133–46.
19.
ChenRTilleyMRWeiHZhouFZhouFMChingS, and others. 2006. Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter. Proc Natl Acad Sci U S A103:9333–8.
20.
ChenXChooHHuangXPYangXStoneORothBL, and others. 2015. The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs. ACS Chem Neurosci6:476–84.
21.
ChoJRyuSLeeSKimJKimHI. 2020. Optimizing clozapine for chemogenetic neuromodulation of somatosensory cortex. Sci Rep10:6001.
22.
CopeZAVazeyEMFlorescoSBAston JonesGS. 2019. DREADD-mediated modulation of locus coeruleus inputs to mPFC improves strategy set-shifting. Neurobiol Learn Mem161:1–11.
23.
CourtneyNAMamaligasAAFordCP. 2012. Species differences in somatodendritic dopamine transmission determine D2–autoreceptor-mediated inhibition of ventral tegmental area neuron firing. J Neurosci32:13520–8.
24.
CubedduLXHoffmannIS. 1982. Operational characteristics of the inhibitory feedback mechanism for regulation of dopamine release via presynaptic receptors. J Pharmacol Exp Ther223:497–501.
25.
DesloovereJBoonPLarsenLEGoossensMGDelbekeJCarretteE, and others. 2022. Chemogenetic seizure control with clozapine and the novel ligand JHU37160 outperforms the effects of levetiracetam in the intrahippocampal kainic acid mouse model. Neurotherapeutics19:342–51.
EdwardsHVChristianFBaillieGS. 2012. cAMP: novel concepts in compartmentalised signalling. Semin Cell Dev Biol23:181–90.
28.
FergusonSMEskenaziDIshikawaMWanatMJPhillipsPEMDongY, and others. 2011. Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization. Nat Neurosci14:22–4.
29.
FitzpatrickCMRunegaardAHChristiansenSHHansenNWJørgensenSHMcGirrJC, and others. 2019. Differential effects of chemogenetic inhibition of dopamine and norepinephrine neurons in the mouse 5-choice serial reaction time task. Prog Neuropsychopharmacol Biol Psychiatry90:264–76.
30.
FlorescoSBBlockAETseMTL. 2008. Inactivation of the medial prefrontal cortex of the rat impairs strategy set-shifting, but not reversal learning, using a novel, automated procedure. Behav Brain Res190:85–96.
31.
FrançoisALowSASypekEIChristensenAJSotoudehCBeierKT, and others. 2017. A brainstem–spinal cord inhibitory circuit for mechanical pain modulation by GABA and enkephalins. Neuron93:822–39.e826.
32.
GalvanARaperJHuXParéJFBonaventuraJRichieCT, and others. 2019. Ultrastructural localization of DREADDs in monkeys. Eur J Neurosci50:2801–13.
33.
GetherU. 2000. Uncovering molecular mechanisms involved in activation of G protein–coupled receptors. Endocr Rev21:90–113.
34.
GomezJLBonaventuraJLesniakWMathewsWBSysa-ShahPRodriguezLA, and others. 2017. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science357:503–7.
35.
GoossensMGBoonPWadmanWVan den HauteCBaekelandtVVerstraeteAG, and others. 2021. Long-term chemogenetic suppression of seizures in a multifocal rat model of temporal lobe epilepsy. Epilepsia62:659–70.
36.
GoutaudierRCoizetVCarcenacCCarnicellaS. 2020. Compound 21, a two-edged sword with both DREADD-selective and off-target outcomes in rats. PLoS One15:e0238156.
37.
GrafeLACornfeldALuzSValentinoRBhatnagarS. 2017. Orexins mediate sex differences in the stress response and in cognitive flexibility. Biol Psychiatry81:683–92.
38.
GreenwaldECMehtaSZhangJ. 2018. Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem Rev118:11707–94.
39.
GuanCBXuHTJinMYuanXBPooMM. 2007. Long-range Ca2+ signaling from growth cone to soma mediates reversal of neuronal migration induced by slit-2. Cell129:385–95.
40.
GuettierJMGautamDScarselliMRuizdeAzuaILiJHRosemondE, and others. 2009. A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc Natl Acad Sci U S A106:19197–202.
41.
GurevichVVGurevichEV. 2017. Molecular mechanisms of GPCR signaling: a structural perspective. Int J Mol Sci18:2519.
42.
HallsML. 2019. Localised GPCR signalling as revealed by FRET biosensors. Curr Opin Cell Biol57:48–56.
43.
HamlettEDLedreuxAGilmoreAVazeyEMAston-JonesGBogerHA, and others. 2020. Inhibitory designer receptors aggravate memory loss in a mouse model of down syndrome. Neurobiol Dis134:104616.
44.
HillDBPlazaMJBoninKHolzwarthG. 2004. Fast vesicle transport in PC12 neurites: velocities and forces. Eur Biophys J33:623–32.
45.
IlgAKEnkelTBartschDBahnerF. 2018. Behavioral effects of acute systemic low-dose clozapine in wild-type rats: implications for the use of DREADDs in behavioral neuroscience. Front Behav Neurosci12:173.
46.
JendrykaMPalchaudhuriMUrsuDvan der VeenBLissBKätzelD, and others. 2019. Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice. Sci Rep9:4522.
47.
JongYIHarmonSKO’MalleyKL. 2018. GPCR signalling from within the cell. Br J Pharmacol175:4026–35.
48.
KapurSSeemanP. 2001. Does fast dissociation from the dopamine d(2) receptor explain the action of atypical antipsychotics? A new hypothesis. Am J Psychiatry158:360–69.
49.
KehrWCarlssonALindqvistMMagnussonTAtackC. 1972. Evidence for a receptor-mediated feedback control of striatal tyrosine hydroxylase activity. J Pharm Pharmacol24:744–47.
50.
KhokharJYHenricksAMSullivanE.DKGreenAI. 2018. Unique effects of clozapine: a pharmacological perspective. Adv Pharmacol82:137–62.
51.
KholodenkoBN. 2006. Cell-signalling dynamics in time and space. Nat Rev Mol Cell Biol7:165–76.
52.
KrashesMJShahBPMadaraJCOlsonDPStrochlicDEGarfieldAS, and others. 2014. An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature507:238–42.
53.
KroezeWKShefflerDJRothBL. 2003. G-protein-coupled receptors at a glance. J Cell Sci116:4867–9.
54.
KuharMJRitzMCBojaJW. 1991. The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci14:299–302.
55.
KunselmanJMLottJPuthenveeduMA. 2021. Mechanisms of selective G protein–coupled receptor localization and trafficking. Curr Opin Cell Biol71:158–65.
56.
LaceyMGMercuriNBNorthRA. 1987. Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol392:397–416.
57.
LemosJCFriendDMKaplanARShinJHRubinsteinMKravitzAV, and others. 2016. Enhanced GABA transmission drives bradykinesia following loss of dopamine D2 receptor signaling. Neuron90:824–38.
58.
MacLarenDAABrowneRWShawJKKrishnan RadhakrishnanSKharePEspañaRA, and others. 2016. Clozapine-N-oxide administration produces behavioral effects in Long-Evans rats—implications for designing DREADD experiments. eNeuro3(5):ENEURO.0219-16.2016.
59.
MahlerSVBrodnikZDCoxBMBuchtaWCBentzleyBSQuintanillaJ, and others. 2019. Chemogenetic manipulations of ventral tegmental area dopamine neurons reveal multifaceted roles in cocaine abuse. J Neurosci39:503–18.
60.
MahlerSVVazeyEMBeckleyJTKeistlerCRMcGlincheyEMKauflingJ, and others. 2014. Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking. Nat Neurosci17:577–85.
61.
MaiellaroILohseMJKittelRJCalebiroD. 2016. cAMP signals in Drosophila motor neurons are confined to single synaptic boutons. Cell Rep17:1238–46.
62.
ManvichDFWebsterKAFosterSLFarrellMSRitchieJCPorterJH, and others. 2018. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci Rep8:3840.
63.
ManzanequeJMBrainPFNavarroJF. 2002. Effect of low doses of clozapine on behaviour of isolated and group-housed male mice in the elevated plus-maze test. Prog Neuropsychopharmacol Biol Psychiatry26:349–55.
MaudsleySMartinBLuttrellLM. 2007. G protein–coupled receptor signaling complexity in neuronal tissue: implications for novel therapeutics. Curr Alzheimer Res4:3–19.
66.
MazzoneCMPatiDMichaelidesMDiBertoJFoxJHTiptonG, and others. 2018. Acute engagement of G(q)-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior. Mol Psychiatry23:143–53.
67.
McCuddenCRHainsMDKimpleRJSiderovskiDPWillardFS. 2005. G-protein signaling: back to the future. Cell Mol Life Sci62:551–77.
68.
McOmishCELiraAHanksJBGingrichJA. 2012. Clozapine-induced locomotor suppression is mediated by 5-HT2A receptors in the forebrain. Neuropsychopharmacology37:2747–55.
69.
MercuriNBBonciABernardiG. 1997. Electrophysiological pharmacology of the autoreceptor-mediated responses of dopaminergic cells to antiparkinsonian drugs. Trends Pharmacol Sci18:232–5.
70.
MoormanDEAston-JonesG. 2010. Orexin/hypocretin modulates response of ventral tegmental dopamine neurons to prefrontal activation: diurnal influences. J Neurosci30:15585–99.
71.
MuschampJWDominguezJMSatoSMShenRYHullEM. 2007. A role for hypocretin (orexin) in male sexual behavior. J Neurosci27:2837–45.
72.
NagaiYMiyakawaNTakuwaHHoriYOyamaKJiB, and others. 2020. Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys. Nat Neurosci23:1157–67.
73.
NaganumaFNakamuraTKuroyanagiHTanakaMYoshikawaTYanaiK, and others. 2021. Chemogenetic modulation of histaminergic neurons in the tuberomamillary nucleus alters territorial aggression and wakefulness. Sci Rep11:17935.
74.
NairPCMcKinnonRAMinersJOBastiampillaiT. 2020. Binding of clozapine to the GABA(B) receptor: clinical and structural insights. Mol Psychiatry25:1910–9.
75.
NakajimaKWessJ. 2012. Design and functional characterization of a novel, arrestin-biased designer G protein–coupled receptor. Mol Pharmacol82:575–82.
76.
NentwigTBObrayJDVaughanDTChandlerLJ. 2022. Behavioral and slice electrophysiological assessment of DREADD ligand, deschloroclozapine (DCZ) in rats. Sci Rep12:6595.
77.
OliverRJPurohitDCKharidiaKMMandyamCD. 2019. Transient chemogenetic inhibition of D1-MSNs in the dorsal striatum enhances methamphetamine self-administration. Brain Sci9:330.
78.
PeiYRoganSCYanFRothBL. 2008. Engineered GPCRs as tools to modulate signal transduction. Physiology (Bethesda)23:313–21.
79.
PerezPChavret-ReculonERavassardPBouretS. 2022. Using inhibitory DREADDs to silence LC neurons in monkeys. Brain Sci12:206.
80.
PillaiGBrownNAMcAllisterGMilliganGSeabrookGR. 1998. Human D2 and D4 dopamine receptors couple through betagamma G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system: selective antagonism by L-741,626 and L-745,870 respectively. Neuropharmacology37:983–7.
81.
RaperJMorrisonRDDanielsJSHowellLBachevalierJWichmannT, and others. 2017. Metabolism and distribution of clozapine-N-oxide: implications for nonhuman primate chemogenetics. ACS Chem Neurosci8:1570–6.
82.
RayRSCorcoranAEBrustRDKimJCRichersonGBNattieE, and others. 2011. Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition. Science333:637–42.
83.
RitzMCLambRJGoldbergSRKuharMJ. 1987. Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science237:1219–23.
84.
RoddZAEnglemanEATruittWABurkeARMoloshAIBellRL, and others. 2022. CNO administration increases dopamine and glutamate in the medial prefrontal cortex of Wistar rats: further concerns for the validity of the CNO-activated DREADD procedure. Neuroscience491:176–84.
85.
RossWN. 2012. Understanding calcium waves and sparks in central neurons. Nat Rev Neurosci13:157–68.
86.
RothBLShefflerDJKroezeWK. 2004. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat Rev Drug Discov3:353–9.
87.
RoyS. 2014. Seeing the unseen: the hidden world of slow axonal transport. Neuroscientist20:71–81.
88.
RunegaardAHSorensenATFitzpatrickCMJorgensenSHPetersenAVHansenNW, and others. 2018. Locomotor- and reward-enhancing effects of cocaine are differentially regulated by chemogenetic stimulation of Gi-signaling in dopaminergic neurons. eNeuro5(3):ENEURO.0345-17.2018.
89.
SasakiKSuzukiMMiedaMTsujinoNRothBSakuraiT. 2011. Pharmacogenetic modulation of orexin neurons alters sleep/wakefulness states in mice. PLoS One6:e20360.
90.
SmithKSBucciDJLuikartBWMahlerSV. 2016. DREADDS: use and application in behavioral neuroscience. Behav Neurosc130:137–55.
91.
SmithSJBuchananJOssesLRCharltonMPAugustineGJ. 1993. The spatial distribution of calcium signals in squid presynaptic terminals. J Physiol472:573–93.
Stachniak TevyeJGhoshASternson ScottM. 2014. Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus→midbrain pathway for feeding behavior. Neuron82:797–808.
94.
SternsonSMRothBL. 2014. Chemogenetic tools to interrogate brain functions. Ann Rev Neurosci37:387–407.
95.
SzlachtaMKusmiderMPabianPSolichJKolasaMZurawekD, and others. 2018. Repeated clozapine increases the level of serotonin 5-HT1AR heterodimerization with 5-HT2A or dopamine D2 receptors in the mouse cortex. Front Mol Neurosci11:40.
96.
TanahashiSYamamuraSNakagawaMMotomuraEOkadaM. 2012. Clozapine, but not haloperidol, enhances glial D-serine and L-glutamate release in rat frontal cortex and primary cultured astrocytes. Br J Pharmacol165:1543–55.
97.
ThompsonKJKhajehaliEBradleySJNavarreteJSHuangXPSlocumS, and others. 2018. DREADD agonist 21 is an effective agonist for muscarinic-based DREADDs in vitro and in vivo. ACS Pharmacol Transl Sci1:61–72.
98.
TranFHSpearsSLAhnKJEischAJYunS. 2020. Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or Compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice?Neurosci Lett739:135432.
UnglessMASinghVCrowderTLYakaRRonDBonciA. 2003. Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons. Neuron39:401–7.
101.
UrbanDJZhuHMarcinkiewczCAMichaelidesMOshibuchiHRheaD, and others. 2016. Elucidation of the behavioral program and neuronal network encoded by dorsal raphe serotonergic neurons. Neuropsychopharmacology41:1404–15.
102.
VardyERobinsonJELiCOlsenRHJDiBertoJFGiguerePM, and others. 2015. A new DREADD facilitates the multiplexed chemogenetic interrogation of behavior. Neuron86:936–46.
103.
VargasMEYamagishiYTessier-LavigneMSagastiA. 2015. Live imaging of calcium dynamics during axon degeneration reveals two functionally distinct phases of calcium influx. J Neurosci35:15026–38.
104.
WerkmanTRMcCrearyACKruseCGWadmanWJ. 2011. NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig. Synapse65:814–26.
105.
WestonMKasererTWuAMouravlevACarpenterJCSnowballA, and others. 2019. Olanzapine: a potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics. Sci Adv5:eaaw1567.
106.
WolfMERothRH. 1990. Autoreceptor regulation of dopamine synthesis. Ann N Y Acad Sci604:323–43.
107.
YoshimuraMNishimuraKNishimuraHSonodaSUenoHMotojimaY, and others. 2017. Activation of endogenous arginine vasopressin neurons inhibit food intake: by using a novel transgenic rat line with DREADDs system. Sci Rep7:15728.
108.
ZhanCZhouJFengQZhangJELinSBaoJ, and others. 2013. Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and hypothalamus, respectively. J Neurosci33:3624–32.
109.
ZhaoZDYangWZGaoCFuXZhangWZhouQ, and others. 2017. A hypothalamic circuit that controls body temperature. Proc Natl Acad Sci U S A114:2042–7.
