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Hurdles in Chronic Chemogenetic Studies

The chronic character of chemogenetics has been put forward as one of the assets of the technique, particularly in comparison to optogenetics. Yet, the vast majority of chemogenetic studies have focused on acute applications, while repeated, long-term neuromodulation has only been booming in the past few years. Unfortunately, together with the rising number of studies, various hurdles have also been uncovered, especially in relation to its chronic application. It becomes increasingly clear that chronic neuromodulation warrants caution and that the effects of acute neuromodulation cannot be extrapolated towards chronic experiments. Deciphering the underlying cellular and molecular causes of these discrepancies could truly unlock the chronic chemogenetic toolbox and possibly even pave the way for chemogenetics towards clinical application. 

  • chemogenetics
  • designer receptor activated by designer drugs (DREADD)
  • neuromodulation

1. Introduction

Neurobiology research has undoubtedly been revolutionized following the introduction of opto- and chemogenetics. The breakthrough of targeted neuromodulation started with the introduction of optogenetics by the Deisseroth lab in 2005 and the proclamation of this technique as the “Method of the Year” in 2010 by Nature [1]. Optogenetics finds itself at the intersection of various disciplines, i.e., virology, genetics, biochemistry, and biology. It combines targeted expression of a light-sensitive modulator, via viral vector or transgenic approaches, with photo stimulation—typically achieved via an optical fiber connected to an external laser—to attain targeted control of specific cellular populations in an in vivo setting. A few years later, chemogenetics has been pushed forward as an alternative technique to optogenetics, replacing optics (light sensitive modulators and light stimulation) with pharmacology (drug sensitive modulators and drug stimulation). The use of chemogenetics was spearheaded after the introduction of DREADDs (Designer Receptors Exclusively Activated by a Designer Drug) in 2007 by the Roth lab [2]. As the acronym implies, DREADD is an umbrella term encompassing a group of genetically engineered G protein-coupled receptors (GPCRs) that have an altered ligand responsiveness. DREADDs are unresponsive to their native, endogenous ligands, but are instead exclusively switched on by engineered drugs [3]. For example, the DREADD prototypes hM3Dq (stimulatory) and hM4Di (inhibitory) are no longer activated by acetylcholine, yet hijacked to respond to the drug clozapine-N-oxide (CNO) [3]. Briefly, hM3Dq activation triggers the phospholipase C cascade, causing the release of intracellular calcium and membrane depolarization. On the other hand, hM4Di inhibits the adenylyl cyclase cascade and activates inward rectifying potassium channels, leading to membrane hyperpolarization [2][4][5]. Yet, many other DREADD receptors exist, such as hM3Ds and rM3D; or KORDi, which is activated by salvinorin B instead of CNO [6][7].

2. The DREADD Actuator CNO

A prototypical DREADD experiment includes the use of the archetypal DREADD ligand CNO, yet this also represents one of the most frequently stated critiques on the platform. Evidence suggests that not CNO, but its parent metabolite clozapine, permeates the blood–brain barrier and is the actual DREADD activator in many laboratory animals, including rodents [8][9][10][11]. Clozapine is a therapeutically approved antipsychotic drug that, when present at high levels, binds to a variety of endogenous receptors with well-known effects on animal behavior [12]. Since the majority of reports employ DREADDs in behavioral studies, such off-target effects can easily confound the study results. Nonetheless, low doses of CNO (≤3 mg/kg bodyweight) are reported to result in subthreshold clozapine concentrations that are unlikely to bind with endogenous receptors, as the affinity of clozapine for DREADDs is much higher [9][13][14][15]. Although many studies demonstrated the absence of behavioral off-target effects induced by CNO or back-metabolized clozapine in animals without DREADD expression [16][17][18][19][20][21], other ligands have been developed to overcome this concern—e.g., olanzapine [22], perlapine [23], compound 21 [23], deschloroclozapine [24], and JHU37160/152 [25]. The absence of off-target effects of these new generation of DREADD actuators also remains to be demonstrated. Although the number of studies including other DREADD ligands is rising, CNO is still by far the most used DREADD activator, even in chronic DREADD studies, in spite of all critiques. Whether this is related to the superiority of CNO as DREADD actuator; to its commercial availability; or to the inertia of scientific practice, i.e., the risk-averse option to stick to the most widely used and conventional method, is not clear. This led to the consensus that, regardless of the chosen DREADD actuator, findings of a DREADD study are not discounted when employing a well-considered, rigorous experimental design with proper control experiments and tailored dosing of the DREADD actuator.
One remaining question is whether clozapine should be administered as DREADD actuator instead of CNO. This could avoid variations in CNO-to-clozapine conversion, thus offering a better control of clozapine dosing [9][11]. However, in applications in which prolonged DREADD activation is required, CNO metabolism could offer some advantage as it steers a gradual production of clozapine, possibly extending the time span of neuronal manipulation [9][11]. Yet, repeated CNO administrations could also cause clozapine accumulation, reaching clozapine doses that are too high to avoid non-DREADD related side effects [26]. To draw definite conclusions, this matter should be studied in more detail.
Although DREADDs form the leading chemogenetic platform, other approaches were also developed [6][7][27], such as the pharmacologically selective actuator/effector module (PSAM/PSEM) tool. This platform is not based on GPCR signaling, but instead hijacks ligand-gated ion channels [6][7][28]. Due to the ionotropic mechanism of action, the PSAM/PSEM platform leans more towards optogenetics, also in terms of the timescale of neuromodulation, i.e., ±30 min of neuronal activation upon stimulation [28][29]. Just as CNO, the PSEM ligand suffers from a number of flaws, primarily the need of high concentrations to achieve adequate in vivo efficiency and its short half-life [27][28][30]. Recently, so-called ultrapotent PSEM (or uPSEM in short) ligands were developed [30][31]. These ligands are highly effective at low doses and show great brain penetrance upon systemic administration in both mice and nonhuman primates, therefore showing great promise for future clinical applications.

3. Cell Specificity

The ability to specifically modulate a single cell population whilst leaving the others unaffected offers key benefits to the neuroscience research field and can be accomplished by the DREADD platform. The most popular route to administer the DREADD ligand is via intraperitoneal/subcutaneous injection(s); yet, given the need for repeated ligand administration in chronic experiments, other systemic administration routes were introduced as well. Examples include adding the designer drug to drinking water or food pellets [32][33][34], micropipette-guided oral administration [35], use of eye drops [36], or implanted minipumps [19][37]. Although one could opt for non-systemic, yet more invasive, ligand delivery routes such as the use of intracranial cannulae [38] or magnetoliposomes [39], most chemogenetic studies still apply a systemic and non-invasive administration, which implies that the DREADD construct should be specifically targeted to the cell population of interest. The DREADD construct is typically introduced via vector-mediated delivery with cell-type specific promotors, usually packed within adeno-associated viral vectors (AAVs). Upon diffusion of the vector to connected regions, off-target expression of the chemogenetic modulators can occur, which can be disadvantageous upon systemic administration of the DREADD ligand. Vector diffusion can, however, be limited by optimizing the vector’s serotype, titer, and injection volume [40]. Alternatively, to avoid off-target expression, recombinase strategies such as Cre-Lox, FLP-FRT, or Tet expression systems can be employed [41][42][43][44]. Another option to insert the DREADD modulators into the genome is via DREADD-expressing transgenic mice, with or without recombinase approaches. Currently, there are 19 chemogenetic mouse lines commercially available at the Jackson Laboratory (, accessed on 2 December 2021). Of note, the recombinase strategies can suffer from “leaky” expression, i.e., expression in the absence of the recombinase [45]. This is most certainly troublesome in transgenic mouse lines, in which the DREADD construct could have been inserted in the entire central nervous system and even in peripheral tissues, which makes it fundamentally difficult to exclude the effects of possible leaky expression on the study results. As such, localized viral vector injections still render an additional layer of specificity as compared to transgenic approaches [40].

4. Lack of Fundamental Knowledge of Chronic DREADD Neuromodulation

Despite the fact that DREADDs were introduced more than a decade ago, their chronic use was largely unexplored until the past few years. A major advantage of chronic chemogenetic experiments is that it empowers long-term and longitudinal studies. Given the simplicity and availability of the DREADD platform, researchers adopted this plug-and-play tool in chronic experimental designs without first scrutinizing the underlying cellular and molecular actions of chronic neuromodulation. Skipping the molecular basis of chronic neuromodulation and directly probing its effect on behavioral readouts was a long shot. This is underscored by studies comparing results obtained from acute and chronic DREADD applications. Although some of those experiments show a similar level of neuronal activity or behavioral outcomes [18][46], many others report null or antagonistic effects upon continuous DREADD activation [17][18][19][32][33][34][37][47][48][49][50][51][52][53]. Given these discrepancies, it is clear that there is no straightforward way to extrapolate study results of acute experiments to chronic experiments and more fundamental knowledge of chronic neuromodulation is of key importance. Due to the lack of research into the (molecular) basis of chronic neuromodulation via DREADDs, the exact reasons behind the diverse effects upon chronic DREADD activation remain unclear. Yet, receptor desensitization, feedback mechanisms, as well as neural plasticity have been suggested as contributing, mutually reinforcing factors and are elaborated upon in the sections below.

4.1. Receptor Desensitization

DREADDs are hijacked GPCRs. Endogenous GPCR signaling sets off a secondary messenger chain reaction that amplifies intracellular signals and alters various physiological processes, including the membrane potential and thus neuronal (in)activation [54]. In contrast to a sole and direct altering of membrane potentials via ion channels, as achieved by the PSAM/PSEM platform, DREADD activation thus indirectly affects neuronal activation [40]. It remains unclear how chronically playing with one of the most vital signaling mechanisms of eukaryotic cells will affect the cellular and molecular physiology. On the other hand, it is well-known that overstimulation or constitutively active GPCR signaling can be destructive to the cell [55][56]. Some toxins, such as the cholera toxin, are even recognized to hijack GPCR signaling, causing deleterious permanent G protein activation [57][58]. To keep GPCR signaling within bounds, endogenous GPCRs possess a memory of previous activation. They show a strong tendency to diminish their sensitivity for receptor re-activation after prolonged activation, a phenomenon called receptor desensitization [59][60][61][62]. Furthermore, upon cumulative exposure to stimuli, GPCRs might be downregulated—i.e., internalized and degraded—thereby resulting in a reduced number of receptors on the cell membrane [56][61][62]. Apart from the activation by designer drugs, DREADDs are highly identical to endogenous GPCRs and thus likely to be subjected to receptor desensitization in chronic set-ups [63]. Evidence for receptor desensitization upon repeated DREADD activation can indeed be found in literature. For example, Goossens et al. [37] studied the effects of chronic chemogenetic inhibition of hippocampal neurons in a rat model of temporal lobe epilepsy. Seizure suppression was achieved for the first 4–5 days of treatment, yet not thereafter. The researchers proposed receptor desensitization as a possible mechanism behind this tolerance effect. The occurrence of receptor desensitization was also proposed by Poyraz and colleagues [32], who tried to demonstrate this concept by looking at the effect of an additional acute CNO injection at the end of a 2-week CNO application. Indeed, the additional CNO injection did not affect the behavioral readout; yet, after a 2-day washout period, behavioral effects were reinstated upon acute CNO application. This may suggest that receptor desensitization had occurred, and receptor levels were restored after 48 h of drug abstinence.
More evidence for the existence of receptor desensitization can be found in the employment of either stimulatory or inhibitory DREADDs. The required dose of DREADD actuator is influenced by a number of factors, including the DREADD type [15][64]. Stimulatory DREADDs have a higher efficacy in eliciting neuromodulation as compared to inhibitory DREADDs; as such, the latter require a higher dose of DREADD actuator and are thus more prone to desensitization [41][15][64]. Indeed, all studies reporting desensitization-like effects used inhibitory DREADDs, except for the recent study of Libbrecht et al. [65], who linked receptor desensitization for the first time with stimulatory DREADDs, albeit using a relatively high concentration of CNO (5 mg/kg). Nevertheless, there is ample evidence in literature that chronic chemogenetic experiments with both stimulatory and inhibitory DREADDs do not necessarily lead to desensitization [18][20][46][66][67]. This could potentially be the result of DREADD overexpression, which is in some cases even orders of magnitude higher than endogenous GPCR expression. DREADD overexpression often occurs upon vector-mediated transgene delivery and could instigate receptor reserve, thereby avoiding receptor desensitization [3]. On the other hand, DREADD overexpression is also linked with constitutive activity of the receptor [68][69]. One study reported that DREADD overexpression perturbed endogenous GPCR signaling and alterations in both ion channel activity and intracellular signaling in the absence of the DREADD ligand [68]. Various other studies examining this concept did not report constitutive DREADD activity, yet when moving to clinical applications, researchers should invest in studying the consequences of lifelong DREADD overexpression [69]. In summary, the occurrence of receptor desensitization again advocates for thought-out dosing and administration schemes of DREADD ligands in chronic paradigms, as well as more fundamental research into the phenomenon of receptor desensitization and overexpression.

4.2. Neuroadaptive Changes

Plasticity is highly regulated in the adult mammalian central nervous system, for example by the excitatory–inhibitory balance upon enduring network alterations [70]. An interesting detail is that endogenous GPCRs are known to play a key role in synaptic and structural plasticity, as well as in activity-related plastic phenomena such as long-term potentiation or depression [71][72][73][74]. As such, it is not surprising that continuous neuronal stimulation/inhibition via DREADDs could be accompanied by plastic events and lead to compensatory responses, which could explain the paradoxical outcomes in acute versus chronic DREADD experiments [70]. The involvement of plasticity in DREADD activation is supported by studies that report long-lasting behavioral and physiological effects that persist over time (up to 1 month) after discontinuation of chronic CNO treatment [20][75][76]. For example, Pozhidayeva et al. [20] studied binge-like drinking behavior in mice upon chronic administration of CNO in combination with both stimulatory or inhibitory DREADDs in the nucleus accumbens. Chronic CNO application reduced alcohol consumption and this effect lasted up to 1 week after discontinuation of chronic treatment. The researchers reported changes in neuronal morphology potentially induced by plastic events, as well as changes in the expression profile of plasticity-related genes. Furthermore, Salesse et al. [75] chronically inhibited dopaminergic circuits in postnatal mice using DREADDs and noted that the observed increase in locomotor activity and stereotypic behavior was still present 1 month after cessation of CNO injections. Moreover, Xie et al. [76] revealed that cardiovascular dysfunction was still present 2 to 3 days after the last CNO injection in a study in which they chronically activated glial cells in the murine sympathetic ganglia via DREADDs. Interestingly, rebound effects after cessation of chronic DREADD treatments are observed as well, again hinting towards network alterations or compensations due to prolonged treatment. For example, Desloovere et al. [12] showed a suppression of epileptic seizures in a mouse model for temporal lobe epilepsy upon chronic use of inhibitory DREADDs for 3 days. Yet, 1 day after withdrawal of clozapine injections, the fraction of time spent in seizures was significantly higher and even exceeded baseline levels. A last example of adaptive changes upon chronic chemogenetic modulation is the study of Binning et al. [49]. The researchers show that repetitive stimulation of microglia for 4 consecutive days instigated microglial memory formation, thereby priming these cells for future neuroinflammatory events. Indeed, after chronic microglial activation, a decreased inflammatory response was observed upon lipopolysaccharide-induced inflammation. Hence, a deeper understanding of neuroadaptive changes in chronic DREADD applications is required.

This entry is adapted from 10.3390/cells11071110


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