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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Feb 3;177(5):992–1002. doi: 10.1111/bph.14885

Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications

Lore M Peeters 1, Stephan Missault 1, Aneta J Keliris 1, Georgios A Keliris 1,✉
PMCID: PMC7042113  PMID: 31658365

Abstract

The combination of chemogenetics targeting specific brain cell populations with in vivo imaging techniques provides scientists with a powerful new tool to study functional neural networks at the whole‐brain scale. A number of recent studies indicate the potential of this approach to increase our understanding of brain function in health and disease. In this review, we discuss the employment of a specific chemogenetic tool, designer receptors exclusively activated by designer drugs, in conjunction with non‐invasive neuroimaging techniques such as PET and MRI. We highlight the utility of using this multiscale approach in longitudinal studies and its ability to identify novel brain circuits relevant to behaviour that can be monitored in parallel. In addition, some identified shortcomings in this technique and more recent efforts to overcome them are also presented. Finally, we discuss the translational potential of designer receptors exclusively activated by designer drugs in neuroimaging and the promise it holds for future neurotheranostic applications.

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Abbreviations

AAV

adeno‐associated virus

ASL‐MRI

arterial spin‐labelling MRI

BBB

blood–brain barrier

BOLD

blood oxygenation level dependent

C13

compound 13

C21

compound 21

C22

compound 22

CBF

cerebral blood flow

CNO

clozapine‐N‐oxide

DREADDs

designer receptors exclusively activated by designer drugs

DREAMM

DREADD‐assisted metabolic mapping

FDG

fluorodeoxyglucose

KORD

κ opioid receptor based DREADD

Pdyn

prodynorphin

Penk

proenkephalin

PSAM

pharmacologically selective actuator module

PSEM

pharmacologically selective effector molecule

SalB

salvinorin B

1. INTRODUCTION

In order to understand brain function in health and disease, it is essential to perform specific spatiotemporal manipulations of neuronal circuits and cellular signalling. During the last two decades, scientists have developed an excellent array of genetically based tools that allowed investigators to selectively control the activity of neuronal cell populations and thereby increase our understanding of the network dynamics in the CNS (Deisseroth, 2015; Roth, 2016). Apart from the better established optogenetic tools, various chemogenetic approaches are gaining relevance for neuroscientific and therapeutic applications (Magnus et al., 2019; Roth, 2016). Chemogenetics as used here, refers to modulatory tools that utilise engineered receptors such as designer receptors exclusively activated by designer drugs (DREADDs) to reversibly control cell populations through systemic administration or microinfusion of a biologically inert ligand. Compared with optogenetics, chemogenetics eliminates the necessity for light and the implantation of an optic fibre to control cell activity. Although the temporal precision and control of chemogenetics is lower than optogenetics, it has been proven to be an invaluable tool in modern neuroscience allowing cell‐type‐specific modulations, bidirectional control of cells, and mapping of functional networks. While there are several recent excellent reviews reporting on the genetic/molecular aspects of DREADDs‐mediated modulations (Roth, 2016; Urban & Roth, 2015), here, we will focus on their utility for neuroimaging‐theranostic applications involving PET and MRI. Theranostics refers to a rapidly growing area of research that combines specific targeted therapy and diagnostics in order to allow future personalised and effective medicine approaches. Neurotheranostics is a new subfield of theranostics directed specifically towards treatment and imaging of brain disorders. In this line, the use of chemogenetics along with PET and MRI would have a marked effect on the treatment of brain disorders by allowing the control and/or repair of neuronal circuits (by DREADDs) related to neurological diseases with non‐invasive mapping of the related functional changes and dynamics across the whole brain (by PET and MRI; Figure 1).

Figure 1.

Figure 1

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Schematic overview of the DREADD technology and its combination with imaging techniques, PET and MRI. (a) DNA coding for the DREADD (hM3Dq or hM4Di) and a fluorescent tag are inserted into a viral vector. Then, this virus is stereotactically injected into a target brain area. Here, the transfected cells will express the DREADD and fluorescent tag. DREADDs activation can be achieved by, for example, systemic injection of a DREADDs agonist. (b) Binding of a DREADDs agonist to the DREADDs results in DREADDs activation which can induce neuronal inhibition (no action potentials) or neuronal activation (increased number of action potentials). (c) Combining the DREADDs technology with PET and MRI allows the non‐invasive evaluation of receptor occupancy and the effect of selective activation/inhibition of a specific neuronal population on large‐scale networks, respectively

1.1. DREADDs development and characterisation

The first class of DREADDs to be introduced were modified human https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=2 that were engineered to be insensitive to their endogenous agonist (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294) by the introduction of two mutations, that is, Y149C3.33 and A239G5.46. Instead, they can be activated by pharmacologically inert ligands (DREADD activators) that demonstrate high specificity and affinity (Alexander et al., 2009). A battery of different muscarinic‐based DREADDs exert their functions through three main G‐protein signalling pathways, namely, Gq, Gi, and Gs. The Gq DREADDs induce increased neuronal activity via stimulation of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=274 and consequent release of intracellular calcium. The Gi DREADDs mediate neuronal silencing via inhibition of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=257 activity, resulting in decreased production of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2352, an important second messenger, and/or stimulation of https://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=74 and inhibition of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=80 channels, which hyperpolarises the neuronal membrane. Activation of the less commonly used Gs DREADDs induces enhanced production of cAMP leading to increased cell activity (Armbruster, Li, Pausch, Herlitze, & Roth, 2007; Conklin et al., 2008; Urban & Roth, 2015).

In vivo, DREADDs expression in specific cell types is typically achieved by using transgenic mouse models or focal viral vector injections targeting selected brain areas. Various transgenic mouse models have been developed to specifically express DREADDs in selected cell populations by using specific promoters (Farrell et al., 2013; Guettier et al., 2009), the transcriptional Tet‐On/Off system (Alexander et al., 2009) or the Cre‐recombinase system (Zhu et al., 2016). Transgenic lines lower the inter‐animal variability by offering similar expression levels of DREADDs across all animals, while losing the ability to non‐invasively manipulate the receptors in a region‐specific manner. On the other hand, DREADDs can also be expressed using a virally mediated method. In this case, a viral construct is intracranially injected and the cellular transduction and expression of the DREADDs is determined by the viral vector's serotype (of genome and capsid) and promoter gene. The most commonly used vectors are recombinant adeno‐associated viruses (AAVs). Different serotypes of AAVs demonstrate brain area‐specific differences in transduction and retrograde transport efficiencies (Aschauer, Kreuz, & Rumpel, 2013). Other viral vectors, such as lentiviral and herpes simplex viral vectors, have also been used to express DREADDs in vivo (Eldridge et al., 2016; Ferguson et al., 2011). Furthermore, various promotors including calmodulin kinase IIa (Alexander et al., 2009), human synapsin (Robinson et al., 2014), glial fibrillary acidic protein (Agulhon et al., 2013), and dynorphin and enkephalin promoters (Ferguson et al., 2011) have been fused with DREADDs to allow cell‐type‐specific expression. These local injections of the viral constructs allow spatial control of the DREADDs expression. Furthermore, the production of functional DREADDs can also be generated by viral constructs encoding DREADDs that are dependent on genetic recombination, such as Cre‐dependent AAVs. The latter restricts the DREADDs to cells that express Cre‐recombinase. Additionally, projection‐specific DREADDs expression can be obtained using Cre‐recombinase expressing canine adenovirus. This Cre‐recombinase expressing canine adenovirus is preferentially retrogradely transported to the neuronal somas, where it can interact with the Cre‐dependent DREADDs constructs to express the DREADDs (Boender et al., 2014; Foldi, Milton, & Oldfield, 2017; Roelofs et al., 2017).

After successful expression of the designer receptors, they can be activated by administration of a designer drug, with clozapine‐N‐oxide (CNO) being the first developed and used since the appearance of DREADDs (CNO, Figure 2; Armbruster et al., 2007). For some years, CNO has been used routinely as the only DREADD‐ligand for chemogenetic activation. CNO can be administered via different routes, including systemic injection (Alexander et al., 2009), intracerebral injection (Mahler et al., 2014), intracerebroventricular injection (Nakajima et al., 2016), in food or drinking water (Cassataro et al., 2014), or in eye drops (Keenan, Fernandez, Shumway, Zhao, & Hattar, 2017). Several studies found that the effects of CNO became apparent after 5–10 min, peaked after 45–50 min and lasted for at least 1–6 hr (Alexander et al., 2009; Roelofs et al., 2017; Vardy et al., 2015). Given this activation time course, the DREADD technology is usually preferred over optogenetics when neuronal activity must be sustainably manipulated over longer time periods.

Figure 2.

Figure 2

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The chemical structure, potency values (EC50/pEC50) and binding affinities (Ki/pKi) of currently used DREADDs agonists: clozapine‐N‐oxide, clozapine, compound 21, JHU37152, JHU37160, JHU37107, perlapine, olanzapine, varenicline, and salvinorin B. References: (1) Bonaventura et al. (2018), (2) Vardy et al. (2015), (3) Thompson et al. (2018), (4) Jendryka et al. (2019), (5) Magnus et al. (2019), (6) Weston et al. (2019)

While CNO has been repeatedly used in numerous studies, recent evidence demonstrated that CNO suffers from its limited ability to cross the blood–brain barrier (BBB) and exerts its effects by conversion to https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=38 (Figure 2), an atypical antipsychotic drug with numerous endogenous receptors (Chang et al., 1998; Gomez et al., 2017; Jann, Lam, & Chang, 1994; Raper et al., 2017). Furthermore, non‐specific, unrelated to DREADDs, effects of CNO have also been demonstrated. Specifically, applying even a low dose of CNO lowered the acoustic startle reflex in rats, while a high dose attenuated the amphetamine‐induced hyperlocomotion in the absence of DREADDs (MacLaren et al., 2016). Given the aforementioned unfavourable characteristics of CNO, recent efforts led to screening of several novel compounds that have been suggested as potent DREADD‐ligands that could replace CNO (Chen et al., 2015). One of the first CNO alternatives, compound 21 (C21, Figure 2), displayed greater selectivity for activating hM3Dq than the native muscarinic receptors (Chen et al., 2015) and has been claimed to be an effective agonist for muscarinic‐based DREADDs both in vitro and in vivo (Thompson et al., 2018). However, Bonaventura et al. recently demonstrated that C21 has low affinity and potency towards DREADDs in vivo and poor BBB penetrance (Bonaventura et al., 2018). Further, the same authors investigated other compounds from the series published originally by Chen et al. (2015), more specifically compound 13 (C13) and compound 22 (C22), and suggested them as more promising DREADD agonists. Michaelides et al. made a great step forward in the field of chemogenetics by developing a new generation of specific and efficient DREADD‐ligands, JHU37152 (Figure 2) and JHU37160 (Figure 2), showing good BBB permeability and exhibiting high in vivo DREADD potency in mice, rats, and macaques, underlining their translational potential (Bonaventura et al., 2018). Data from our group also suggest that the novel DREADD agonist JHU37160 is much more potent than CNO. Our data show that injection of 0.1 mg·kg−1 JHU37160 induces a more pronounced and widespread increase in blood oxygenation level dependent (BOLD) signal compared to 1 mg·kg−1 CNO. Furthermore, JHU37160 seemed to induce a more consistent response in both subjects than CNO, which might be explained by the difference between the animals in their ability to convert CNO to clozapine (Figure 3). Other compounds that might be suitable for activating DREADDs are perlapine (Chen et al., 2015) and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=47 (Weston et al., 2019; Figure 2).

Figure 3.

Figure 3

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(a) DREADDs‐induced activation using pharmacological functional MRI. Single‐subject statistical difference maps of blood oxygen level‐dependent (BOLD) signal after DREADD agonist administration (FWE corrected, P < .05, minimal cluster size k ≥ 10). The colour scale indicates t‐values. Two male adult Long–Evans rats expressing hM3Dq‐DREADDs in the right somatosensory cortex (site of injection indicated by black dot and arrow) were subjected to pharmacological fMRI to assess the BOLD response after intravenous administration of the DREADD agonists JHU37160 and CNO. Data were processed according to Missault et al., (2019) with a few modifications (see Supporting Information). Both DREADD agonists resulted in an increased BOLD response, reflecting increased neuronal activity. However, JHU37160 induced a more pronounced and widespread response compared with CNO in the same subjects. JHU37160 also seemed to induce a more consistent response in both subjects than CNO, which might be explained by a difference between the animals in their ability to convert CNO to clozapine. The doses of JHU37160 and CNO were chosen based on the average doses reported in literature. (b) Visualisation of DREADDs using PET imaging. In the study by Gomez et al. (2017) rats were injected with GFP‐expressing adeno‐associated virus (AAV) in the left hemisphere and hM4Di‐mCherry‐expressing AAV in the right hemisphere. PET imaging was performed with [11C]clozapine. Highly localised [11C]clozapine binding (co‐registered to a MRI rat template) was observed at the injection site of the hM4Di‐mCherry‐expressing AAV (B3), which colocalised with both hM4Di‐mCherry expression (B1) and the [3H]clozapine autoradiography signal (B2). Modified from Gomez et al., (2017): reprinted with permission from AAAS

Although the development of new second‐generation DREADD agonists is progressing, one main drawback remains, namely, all excitatory and inhibitory muscarinic‐based DREADDs are sensitive to the same ligands. To this end, Vardy et al. (2015) developed an inhibitory DREADD based on a Gi‐coupled https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=318 (KORD) that selectively activates upon binding of the inert ligand salvinorin B (SalB; Vardy et al., 2015). SalB is an inactive metabolite of the κ opioid receptor‐selective agonist salvinorin A and has desirable pharmacokinetic properties and BBB permeability (Hooker et al., 2009). Several studies demonstrated KORD‐mediated behavioural alterations that peaked at 10 min after subcutaneous SalB administration and which last up to 1 hr in rodents (Marchant et al., 2016; Marchant et al., 2016; Vardy et al., 2015). The availability of these new designer receptors allows bidirectional control of a cellular population by co‐expression of excitatory muscarinic‐based DREADDs and KORD in the same cells (Vardy et al., 2015).

In an effort to get closer to neurotheranostic applications, Magnus et al. have recently designed a pharmacologically selective actuator module (PSAM), that is, PSAM4, for which the clinically approved smoking cessation drug https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5459 has a very high affinity (Figure 2). In addition, they designed ultrapotent pharmacologically selective effector molecules with even higher affinity for this PSAM4 and which are more selective than varenicline. In combination with different ion pore domains (e.g., https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=68 or https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=73), PSAM4 produces chimeric ligand‐gated ion channels, which allow direct control of cellular activity. Varenicline and the different newly developed ultrapotent pharmacologically selective effector molecules (PSEMs) had long‐lasting effects (several hours) in mice. Thus, these newly developed PSAM and PSEMs are also very promising chemogenetic tools and hold great translational potential (Magnus et al., 2019).

Neurotherapeutics would benefit from new technologies that allow non‐invasive on‐demand control of the activity of specific cells. The DREADDs technology as well as the PSAM4 technology seem to be good candidates for clinical applications. The translational potential of DREADDs has already been suggested by its use in several non‐human primate studies (Eldridge et al., 2016; Grayson et al., 2016; Nagai et al., 2016; Raper et al., 2017; see Section 2 for a detailed discussion). Moreover, AAV technology has proven safe and promising in several clinical studies such as those targeting coagulation disorders, inherited blindness, and Parkinson's disease (Christine et al., 2009; Colella, Ronzitti, & Mingozzi, 2018; Nathwani et al., 2011; Pierce & Bennett, 2015). Furthermore, recent efforts have focused on the development of a non‐invasive method for efficient gene delivery to the CNS using intravenous AAVs injections (Chan et al., 2017).

2. DREADDS COMBINED WITH IMAGING TECHNIQUES

Although DREADDs have demonstrated great potential for neural manipulation and control, their overall effects on large‐scale neural networks and the mechanisms of action at brain scales relevant to behaviour remain poorly understood. Thus, complementing this powerful approach with non‐invasive readout techniques that can uncover DREADDs expression and localisation in vivo as well as their functional activity profiles and manipulation of neural network patterns would be a major advance. Some first efforts towards this endeavour have combined DREADDs with neuroimaging technologies such as PET and MRI. Both modalities are extensively used in functional brain imaging that typically utilises the alterations in metabolism (e.g., cerebral blood flow [CBF], cerebral blood volume, oxygen, or glucose metabolism) or neurochemical processes (e.g., receptor occupancy) to assess regional changes in neuronal activity. PET imaging captures neuronal activity via measuring the levels of “radioactive tracers,” such as 18F‐fluorodeoxyglucose (18F‐FDG), which are intravenously injected and transported via the blood into active areas. Functional MRI provides an indirect measure of neuronal activity by detecting changes in blood oxygenation and blood flow. While PET and functional MRI have been extensively used to measure brain activity, each of them have their own advantages and disadvantages. For instance, PET enables high‐sensitivity mapping and quantification of target molecules such as DREADDs using radioactive ligands albeit with low spatial resolution. On the other hand, functional MRI provides images with high spatial and temporal resolution without the use of ionising radiation, but is limited by the low detection sensitivity. Hence, the use of multimodal PET and functional MRI provides complementary means to follow chemogenetic manipulations of neuronal activity in translational and basic research. Of note, hybrid PET–MRI scanners are gaining attention and might expand frontiers for neurotheranostic applications with DREADDs. Here, we briefly present current directions towards such neurotheranostic applications with DREADDs.

2.1. Imaging DREADDs expression and the quest for the ideal DREADDs agonist

While expression of DREADDs is typically confirmed ex vivo, the use of non‐invasive techniques allowing the assessment of successful transfection in vivo prior to experiments can substantially increase the translational potential of DREADDs and reduce the cost and variability of in vivo studies. For example, DREADDs expression can be visualised non‐invasively by highly sensitive PET imaging using radiolabelled DREADDs agonists. Moreover, longitudinal follow‐up of expression patterns is necessary given that, according to some studies, desensitisation and receptor internalisation leading to down‐regulation over time can occur upon frequent and repeated activation of DREADDs. Several DREADDs agonists have been radiolabelled for the purpose of imaging DREADDs in vivo. Ji et al. (2016) compared the in vivo performance of [11C]CNO and [11C]clozapine for visualising inhibitory DREADDs (hM4Di). They concluded that [11C]CNO was the better choice for high‐contrast imaging of hM4Di in mice with minimal background signal. Intravenous administration of [11C]clozapine resulted in modest radioactivity retention in mice that were not expressing any DREADDs, which was not the case for [11C]CNO. This result is thought to be due to the higher affinity of clozapine for endogenous receptors, compared to CNO. Hence, it was easier to distinguish between regions with and without hM4Di expression with [11C]CNO than [11C]clozapine. However, the authors reported that the overall lower uptake of [11C]CNO in the brain might limit its sensitivity in detecting hM4Di, especially if the sensitivity of the PET imaging device or the injected radioactivity dose are limited. In addition, a pilot study by this research group indicated that [11C]clozapine might be better than [11C]CNO for visualising hM4Di expression in the brain of rhesus monkeys (see also below or Nagai et al., 2016). [11C]CNO and [11C]clozapine displayed different dynamic profiles in the mouse study. The continuous increase in radioactivity retention in the target area (expressing hM4Di) over 1 hr following intravenous administration of [11C]CNO may be the result of binding of radioactive metabolites of [11C]CNO such as [11C]clozapine. Ji et al. used hM4Di as a PET‐visible reporter to obtain long‐term monitoring of the differentiation of induced pluripotent stem cell‐derived implants to mature neurons in mice. Moreover, DREADDs allow reversible modulation of the activity of the grafts and the connecting host neurons. hM4Di‐induced pluripotent stem cells might be a more beneficial replacement therapy than viral vector‐mediated gene transfer to surviving cells, especially in neurodegenerative disorders where there is major neuronal loss (Ji et al., 2016).

Nagai et al. (2016) used [11C]clozapine to perform PET imaging of hM4Di in macaque monkeys. PET scans with [11C]clozapine were used to confirm successful expression of hM4Di in the target region following stereotaxic injection of a viral vector. While there was some background signal (probably due to the affinity of [11C]clozapine for innate receptors), radiotracer uptake at hM4Di‐expressing regions was ~20% higher than in non‐hM4Di‐expressing regions. Weak or mistargeted DREADD expression explained the lack of a behavioural effect of DREADD activation with CNO. PET scans with [11C]clozapine in monkeys pretreated with increasing doses of CNO were used to determine the optimal dose of CNO for the behavioural experiments. A CNO dose of 3 mg·kg−1 was chosen since this resulted in a 50–60% occupancy of the DREADDs. Repetitive (once weekly) treatment with CNO did not result in a significant decrease in DREADDs at the target site, as shown by PET scans with [11C]clozapine. This study demonstrated that repeated inactivation of the rostromedial caudate results in a loss of sensitivity to reward value in the decision making of monkeys (Nagai et al., 2016).

Gomez et al. (2017) compared [11C]CNO and [11C]clozapine for PET imaging of hM4Di in rats injected with AAV‐hM4Di and observed no radiotracer uptake with [11C]CNO, but high radiotracer binding and specific hM4Di occupancy after intravenous [11C]clozapine administration. They also did not observe any radioligand uptake in the brains of DREADD‐expressing mice after systemic [3H]CNO injection, but detected high brain uptake and DREADD‐specific binding following systemic [3H]clozapine administration. This led the authors to conclude that CNO cannot enter the brain after systemic injection as discussed earlier (Section 1). They observed that [11C]clozapine accounted for 31% of the total radioactivity in the brain of hM4Di‐expressing rats after intravenous injection of [11C]CNO, while [11C]CNO accounted for 44% of the total radioactivity. In control rats, no [11C]clozapine was observed in the brain after intravenous injection of [11C]CNO, while [11C]CNO accounted for 76% of the total radioactivity. Detection of [11C]CNO in the brain samples was ascribed to [11C]CNO present in the blood vessels of the brain or in the CSF, but not within the brain parenchyma. As discussed in Section 1, CNO displayed low affinity for DREADDs in in vitro assays. CNO rapidly converts to clozapine in vivo, which readily enters the brain and has high affinity for and potency at DREADDs. Low doses of clozapine (0.1 mg·kg−1 in rats and 0.01 mg·kg−1 in mice) induced preferential DREADD‐mediated behaviours but had no effect in animals lacking DREADDs. Much higher doses of CNO (10 mg·kg−1, 100‐fold higher) were needed to induce the same behaviour in DREADD‐expressing rats, consistent with plasma levels of converted [11C]clozapine (~2%) following systemic [11C]CNO injection. Also, at 10 μM, CNO competitively inhibited binding at several endogenous receptors in an in vitro assay, including histamine https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=262, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=6, muscarinic https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=13, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=15, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=16, and dopamine https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214 and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=215 receptors. Hence, the agonistic effects of CNO at DREADDs could be confounded by off‐target effects at endogenous receptors. Moreover, injection of 1 mg·kg−1 CNO resulted in behavioural alterations at 2–3 hr post‐injection in control mice without DREADDs, when CSF levels of converted clozapine peak. No such effect was observed after injection of 0.01 mg·kg−1 clozapine, at this time‐point or earlier. These late‐onset non‐specific effects of CNO should be taken into consideration when choosing the experimental time frame in CNO experiments. Indeed, based on these observations, it was concluded that researchers should use low doses of clozapine to activate DREADDs instead of high doses of CNO. Moreover, inter‐subject differences in metabolism can increase experimental variability and result in poor reproducibility of findings (Gomez et al., 2017).

Bonaventura et al. (2018) evaluated several novel DREADDs agonists in their search for an 18F‐labelled PET DREADDs radiotracer with high in vivo affinity. Initially, they evaluated the in vivo activity of C21 in DREADDs‐expressing rodents and non‐human primates and concluded it has suboptimal in vivo DREADDs affinity and potency. Compared to clozapine, 100‐ to 1,000‐fold higher doses of C21 were needed to induce DREADDs‐specific behaviours. At higher doses, C21 induced sedative effects in non‐human primates and mice without DREADDs expression. Indeed, unlike clozapine, C21 was shown to bind to https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=319&familyId=50&familyType=GPCR, κ, and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=317 at 10 μM in a competitive binding screen. At doses needed to drive DREADDs‐specific behaviours, no behavioural changes were observed in mice without DREADDs, but significant changes were seen in these mice in brain metabolic activity using [18F]fluorodeoxyglucose (FDG)‐PET. An equipotent dose of clozapine did not produce any such effects. Hence, they concluded that C21 seems to be unsuitable as a DREADDs agonist. In the search for a 18F‐labelled PET DREADDs radioligand, Bonaventura et al. then synthesised fluorinated analogues of C13 and C22 that have been shown to exhibit high DREADDs activity and selectivity in comparison to clozapine (Bonaventura et al., 2018). Three analogues with very high DREADD affinity were identified: JHU37107, JHU37152, and JHU37160 (Figure 2). JHU37152 and JHU37160 have (a) excellent selectivity for DREADDs, (b) high brain penetrability, and (c) high in vivo DREADD affinity and potency. In rodents, both compounds were able to induce DREADDs activation at much lower concentrations in comparison to clozapine (as low as 0.01 mg·kg−1). All three compounds were used to develop 18F‐labelled PET radioligands. Of the three imaging probes, [18F]JHU37107 was the most radiochemically favourable structure, which was radiolabelled with high yield, molar activity, and radiochemical purity. [18F]JHU37107 displayed high in vivo DREADDs binding and allowed visualisation of DREADDs at both the injection site and at proximal and distal projection sites. The radiotracer displayed good pharmacokinetic properties and metabolite profile in a non‐human primate. Thus, [18F]JHU37107 is the first, high‐affinity 18F‐labelled DREADDs PET radioligand, which is able to visualise DREADDs expression at both local and long‐range projection sites (Bonaventura et al., 2018).

Magnus et al. demonstrated that the ligand‐gated ion channel PSAM4‐GlyR could be visualised in mice with the PET radioligand [18F]ASEM, which is already used in the clinic, thereby highlighting its translational potential (Magnus et al., 2019).

2.2. DREADDs in action

Beyond visualising DREADDs expression and estimating potency and affinity as discussed, several studies that have used PET and MRI to assess the whole‐brain response to excitatory or inhibitory chemogenetic activation are discussed below.

2.2.1. DREADD‐assisted metabolic mapping and beyond

Many studies have investigated the effect of selectively activating or inhibiting a set of neurons using DREADDs, on whole‐brain metabolism, more specifically glucose utilisation, using the radiolabelled glucose analogue [18F]FDG. This technique was termed DREADD‐assisted metabolic mapping (DREAMM). The major advantage of this technique is that information can be obtained about cell‐type‐specific functional circuitry in awake, freely moving, behaving animals. Michaelides et al. introduced this technique in 2013 in rats where they targeted prodynorphin‐expressing (Pdyn) and proenkephalin‐expressing (Penk) medium spiny neurons in the nucleus accumbens shell. Selective inhibition of hM4Di‐expressing Pdyn‐ or Penk‐medium spiny neurons using CNO in anaesthetised rats resulted in discrete dynamic metabolic responses (i.e., altered [18F]FDG uptake) in the brain and revealed different network patterns for each of these medium spiny neuron subtypes. The metabolic responses to selective inhibition of Pdyn‐ or Penk‐medium spiny neurons during the awake, behaving state were distinctly different from those observed in anaesthetised rats (Michaelides et al., 2013). Hence, these studies demonstrated that DREAMM can quantitatively assess dynamic whole‐brain changes in cell‐type‐specific functional circuitry activity with single‐minute resolution in anaesthetised rats and provide quantitative whole‐brain information about the neuronal circuits that are recruited in awake, behaving animals upon manipulation of specific neurons. DREAMM is the first technique to allow non‐invasive, longitudinal, quantitative, dynamic whole‐brain mapping of cell‐type‐specific functional circuitry in the awake, freely moving state, based on a direct process of cellular function (i.e., glucose utilisation; Michaelides & Hurd, 2015). Although all PET measurements in these studies were performed in anaesthetised animals, the advent of awake rat PET (Miranda et al., 2019; Schulz et al., 2011) makes it possible to simultaneously measure DREADD‐mediated metabolic changes during behavioural monitoring in awake rats in future studies. Several other studies have made use of DREAMM. Anderson et al. (2013) demonstrated that selective inhibition of hM4Di‐expressing Pdyn neurons in the periamygdaloid cortex of rats using CNO increased the metabolic activity in the extended amygdala. This manipulation also resulted in negative affect‐related physiological and behavioural changes, indicating an important role for impaired Pdyn‐expressing neurons in the periamygdaloid cortex in opiate abuse (Anderson, Michaelides et al., 2013). Urban et al. (2016) demonstrated that acute or chronic selective activation of hM3Dq‐expressing dorsal raphe nucleus serotonergic neurons with CNO resulted in distinct metabolic brain responses and behaviours. Moreover, using DREAMM and slice electrophysiology, they observed that hM3Dq exhibited robust and continuous activity after long‐term (3 weeks) stimulation with CNO (Urban, Zhu et al., 2016). Most recently, Bonaventura et al. (2018) demonstrated with DREAMM that JHU37160‐mediated activation of hM3Dq‐ or hM4Di‐expressing dopaminergic D1 neurons resulted in decreased or increased metabolism in distinct, largely non‐overlapping brain networks, respectively (Bonaventura et al., 2018).

Apart from assessing whole‐brain cellular metabolic activity changes in response to chemogenetic manipulation of specific neurons, PET imaging can also be used to investigate neurotransmitter dynamics when combined with suitable PET tracers, that is, radioligands that bind to neurotransmitter receptors (Michaelides & Hurd, 2015). Lippert et al. (2019) studied the dynamics of dopaminergic signalling in transgenic mice expressing hM3Dq in dopaminergic neurons using PET imaging with [11C]https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=94, which binds to D2 receptors. They reported that low‐frequency variations of extracellular https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=940 concentrations, assessed via fast‐scan cyclic voltammetry, cause detectable temporal variations in the [11C]raclopride PET signal upon chemogenetic activation of dopaminergic neurons using CNO (Lippert et al., 2019).

2.2.2. DREADDs combined with MRI

Some studies have used MRI to capture the effect of selective activation of certain neuronal populations using DREADDs. Baslow et al. (2016) used functional magnetic resonance spectroscopy to evaluate alterations in local neuronal N‐acetylaspartate as well as a BOLD functional MRI to assess focal changes in neuronal activity. In this study, a viral delivery method was used to express hM3Dq in medial prefrontal cortex neurons. CNO injection elicited a focal decrease in the N‐acetylaspartate signal and an increased BOLD response. In addition, local field potentials were recorded as a direct measure of neural activity and confirmed that CNO could increase neural activity through activation of hM3Dq, supporting the utility of DREADDs in vivo (Baslow et al., 2016).

Besides the assessment of local neural activity changes discussed above, MRI is essential for the evaluation of DREADD‐induced effects across the whole brain. Ji et al. (2016) used arterial spin‐labelling MRI (ASL‐MRI) to evaluate DREADD‐induced alterations of neural activity. The CNO‐induced CBF decreases observed coincided with the spatial extent of the hM4Di expression in hM4Di expressing transgenic mice, and no significant CBF alterations were observed in a control region lacking DREADDs or in wild type mice. Furthermore, in another study, ASL‐MRI was used to evaluate the functionality of hM4Di‐expressing neuronal grafts differentiated from induced pluripotent stem cells in wild type mice. The CBF was significantly suppressed at the implantation site upon CNO administration, whereas the CBF remained constant in other brain areas (Ji et al., 2016). In a proof‐of‐concept study on the mesocorticolimbic system in rats, Roelofs et al. expressed hM3Dq receptors in the mesolimbic and mesocortical pathway in a projection‐specific manner. To this end, the DREADDs were expressed in neurons projecting from the ventral tegmental area to the nucleus accumbens or to the medial prefrontal cortex, respectively. BOLD functional MRI was used to identify the effect of stimulation of these neural projections on whole‐brain neural activity. As a result, significantly increased BOLD responses were observed in the ventral tegmental area, nucleus accumbens, and medial prefrontal cortex after CNO injection. Furthermore, the magnitude of the CNO‐induced BOLD response in the ventral tegmental area positively correlated with the number of cFos‐positive neurons in this region. CNO‐induced hyperlocomotion was observed in the animals with DREADDs in the mesolimbic pathway, the time course of which corresponded to the brain activation time course. DREADDs stimulation not only induced activation in the mesocorticolimbic regions but also drove BOLD response alterations at the whole‐brain level. In contrast to the altered neural activity, the functional connectivity remained stable after CNO injection (Roelofs et al., 2017). Although the authors reasoned that neural network activity and functional connectivity are not closely related, care must be taken when interpreting the lack of functional connectivity changes. The rats were anaesthetised with 1.5% https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2505 during the acquisition of the resting‐state functional MRI scans, but a recent study indicated that this dose of isoflurane heavily masks the naturally occurring functional connectivity in the rat brain (Paasonen, Stenroos, Salo, Kiviniemi, & Grohn, 2018). More recently, our group expressed inhibitory KORD in the anterior cingulate area and used BOLD functional MRI to evaluate its effect on neuronal activity and functional connectivity. We observed that inhibition of the anterior cingulate area, which has been identified as a hub region (i.e., a highly interconnected brain region), resulted in large‐scale neuronal activity modulations as well as functional connectivity alterations (Peeters et al., 2019). Furthermore, other recent studies combined chemogenetics with resting‐state functional MRI in order to evaluate functional connectivity alterations after modulation of the excitation–inhibition balance in the somatosensory cortex (Markicevic et al., 2018) or to assess the effects of selective stimulation of the locus coeruleus with hM3Dq on a large‐scale functional connectivity (Zerbi et al., 2019).

Giorgi et al. (2017) highlighted the advantage of chemogenetics combined with functional MRI to causally link the activity of specific neuronal populations with whole‐brain functional activity. These authors used transgenic mice expressing excitatory DREADDs in serotonergic neurons to map the whole‐brain effect of increased endogenous https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5 release and compared these results with the global brain alterations due to systemic administration of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=928 inhibitor https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7547. The stimulation of endogenous serotonergic neurons upon CNO injection resulted in a significantly increased relative cerebral blood volume in connected regions encompassing corticohippocampal and ventrostriatal regions while the administration of citalopram globally decreased the relative cerebral blood volume (Giorgi et al., 2017). This causal link between manipulation of a specific neuronal population and regional brain activation can be further exploited by the multiplexed use of excitatory and inhibitory DREADDs. As such, Benekareddy et al. (2018) expressed excitatory hM3Dq receptors and inhibitory KORD in the prefrontal cortex to elucidate the circuit mediating social behaviour. CNO‐mediated activation of the prefrontal cortex resulted in decreased sociability. Continuous ASL‐MRI revealed CNO‐induced increased perfusion in the prefrontal cortex and perfusion changes in regions involved in emotional behaviour, including the habenula. These effects on perfusion were attenuated upon injection of SalB, which stimulates the inhibitory KORD. SalB was also able to rescue the social behaviour deficit. The combination of chemogenetics with functional MRI and behavioural assessments allowed the identification of the brain regions whose activity is coregulated with that of the prefrontal cortex and different states of social behaviour, ultimately leading to the identification of a corticohabenular circuit regulating socially directed behaviour (Benekareddy et al., 2018). All these MRI studies were performed in rodents, while chemogenetics‐functional MRI has also been used in rhesus monkeys (Grayson et al., 2016). In this case, inhibitory DREADDs were used to temporarily inactivate the amygdala, and resting‐state functional MRI scans were acquired to assess functional connectivity changes. CNO injection significantly decreased functional connectivity in networks directly involving the amygdala as well as in networks that are indirectly linked to the amygdala (Grayson et al., 2016).

3. CONCLUSIONS AND FUTURE PERSPECTIVES

Although, to date, relatively few studies have demonstrated the combined use of chemogenetics and in vivo imaging, the advantages of this multimodal approach are gradually becoming more obvious and will greatly assist scientists to increase their knowledge of brain networks in health and disease. This integrative multiscale approach allows the fundamental investigation of specific neuronal populations within functional networks at the whole‐brain level and their relevance in certain types of behaviour that can be monitored in parallel. Further, by stimulating or inhibiting neuromodulatory circuits, DREADDs allow pharmacological control of brain states longitudinally during behaviour while in vivo imaging can monitor long‐term effects in brain plasticity and network reorganisation. The use of joint DREADDs‐imaging approach also permits the monitoring of DREADDs expression over time as well as the dose effects on receptors' occupancy and outcomes. Importantly, chemogenetics has very high translational potential for neurotheranostic applications. As we discussed, a class of DREADDs responsive to clinically approved medications has been identified, opening windows for proof‐of‐concept studies. In contrast to optogenetics, chemogenetics allow for remote and longer lasting activation/inhibition of neuronal activity without the need for an optic fibre implantation. in vivo imaging will allow non‐invasive, longitudinal monitoring of disease progression and therapy response in patients that will undergo DREADD‐based neuromodulatory therapy. Because DREADDs can be visualised with PET, successful and correct expression of DREADDs following viral vector injection or implantation of induced pluripotent stem cell‐derived neural progenitors can be confirmed. Finally, development of new classes of DREADDs as well as specific agonists that can allow orthogonal and parallel manipulations of more than one circuit is underway. Two very promising novel DREADDs agonists have recently been made commercially available, that is, JHU37152 and JHU37160. These have shown very high in vivo DREADD affinity and brain penetrability and may pave the way for DREADD technology to find its way to the clinic.

3.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos et al., 2019; Alexander, Fabbro et al., 2019; Alexander, Kelly et al., 2019; Alexander, Mathie et al., 2019).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

Data S1: Supporting Information

Click here for additional data file. (71.4KB, pdf)

ACKNOWLEDGEMENTS

This review was supported by the fund of scientific research Flanders (FWO G048917N) and the University Research Fund of University of Antwerp (BOF DOCPRO FFB150340). Stephan Missault is a postdoctoral fellow of the fund of scientific research Flanders (FWO) (12W1619N). We thank Prof. Dr. Michael Michaelides from the Biobehavioral Imaging and Molecular Neuropsychopharmacology Unit, National Institute on Drug Abuse (NIDA) Intramural Research Program (Baltimore, USA) for providing the hSyn‐HA‐hM3Dq AAV and JHU37160 that were used to produce the results shown in Figure 3a.

Peeters LM, Missault S, Keliris AJ, Keliris GA. Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications. Br J Pharmacol. 2020;177:992–1002. 10.1111/bph.14885

Lore M. Peeters and Stephan Missault have equal contribution to this work.

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