DREADD Receptors: Mechanisms and Techniques

Chemogenetics has transformed neuroscience by enabling precise control over cellular activity using engineered receptors activated by synthetic compounds. One of the most widely used tools in this field is Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which allow researchers to modulate neuronal and non-neuronal function with high specificity.

This technique has advanced the understanding of neural circuits, behavior, and disease mechanisms while offering a less invasive alternative to optogenetics or electrical stimulation. Researchers can selectively activate or inhibit specific cell populations to study their physiological roles.

Receptor Modification Techniques

The development of DREADDs relies on molecular engineering to modify G protein-coupled receptors (GPCRs) while preventing activation by endogenous ligands. This process involves mutating muscarinic acetylcholine receptors, specifically the human M3 and M4 subtypes, to alter their ligand-binding domains. Targeted amino acid substitutions create receptors that no longer respond to acetylcholine but instead bind selectively to synthetic ligands such as clozapine-N-oxide (CNO) or deschloroclozapine (DCZ). These modifications ensure receptor activation is both selective and controllable.

To express these modified receptors in specific cell populations, genetic constructs encoding the engineered GPCRs are introduced using viral vectors or transgenic approaches. Adeno-associated viruses (AAVs) and lentiviruses are commonly used due to their ability to drive stable expression in neurons and other cell types. The choice of viral serotype and promoter elements dictates receptor expression specificity, enabling researchers to target distinct neuronal subtypes or brain regions. For example, the CaMKIIα promoter restricts expression to excitatory neurons, while the GFAP promoter ensures selective expression in astrocytes.

Beyond genetic delivery, receptor trafficking and membrane localization influence the effectiveness of DREADD-based interventions. Proper receptor folding and transport to the plasma membrane are necessary for functional activation. Modifications such as adding trafficking sequences from the Kir2.1 potassium channel or the influenza hemagglutinin (HA) tag improve receptor localization, enhancing synthetic ligand-induced signaling. Additionally, factors like promoter strength and viral vector integration affect the stability of DREADD expression over time, requiring careful optimization for consistent receptor function.

Mechanisms of Activation

DREADD activation depends on engineered receptors responding selectively to synthetic ligands while remaining unresponsive to endogenous neurotransmitters. This specificity is achieved through targeted mutations in the ligand-binding domain, which alter receptor conformation to favor interaction with compounds like CNO or DCZ. Upon ligand binding, the receptor undergoes a conformational shift that engages intracellular signaling proteins, leading to downstream cellular effects.

Once activated, the receptor’s interaction with intracellular effectors dictates physiological outcomes. Gq-coupled DREADDs stimulate phospholipase C (PLC), triggering the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). This cascade increases intracellular calcium release, enhancing neuronal excitability and synaptic transmission. Conversely, Gi-coupled DREADDs inhibit adenylyl cyclase, reducing cyclic adenosine monophosphate (cAMP) levels and suppressing neurotransmitter release.

The pharmacokinetics of synthetic ligands influence DREADD activation efficacy and duration. CNO, a commonly used ligand, has slow blood-brain barrier penetration and is metabolized into clozapine, which can have off-target effects at high concentrations. To address this, newer ligands like DCZ offer improved brain penetrance and reduced off-target activity. Studies show DCZ achieves higher receptor occupancy at lower doses than CNO, leading to more reliable activation with minimal systemic interference.

Types of DREADDs

DREADDs are categorized based on their coupling to distinct G protein signaling pathways, each eliciting specific cellular responses. The three primary types—Gq-coupled, Gi-coupled, and Gs-coupled DREADDs—differ in their downstream effects, making them suitable for diverse experimental applications.

Gq-Coupled

Derived from the M3 muscarinic receptor, Gq-coupled DREADDs activate PLC upon ligand binding, hydrolyzing PIP2 into IP3 and DAG. This cascade releases calcium from intracellular stores, enhancing neuronal excitability and synaptic transmission. In behavioral neuroscience, Gq-DREADDs are used to study excitatory signaling in cognition, emotion, and motor control. For example, research in Nature Neuroscience (2016) demonstrated that activating Gq-DREADDs in the prefrontal cortex enhances working memory in rodents. These receptors are also used to investigate astrocyte-neuron interactions, as astrocytic calcium signaling influences synaptic plasticity.

Gi-Coupled

Based on the M4 muscarinic receptor, Gi-coupled DREADDs inhibit adenylyl cyclase, reducing cAMP levels and suppressing neuronal excitability. This inhibition decreases neurotransmitter release and dampens synaptic activity, making Gi-DREADDs useful for studying inhibitory control in neural circuits. In addiction research, these receptors have been used to suppress dopaminergic signaling in the nucleus accumbens, reducing drug-seeking behavior. A study in Biological Psychiatry (2018) found that Gi-DREADD activation in the ventral tegmental area attenuates cocaine-induced reinstatement. Beyond addiction, Gi-DREADDs have been applied to anxiety, pain modulation, and seizure suppression.

Gs-Coupled

Engineered from the β2-adrenergic receptor, Gs-coupled DREADDs stimulate adenylyl cyclase, increasing cAMP levels and promoting excitatory signaling. Unlike Gq-DREADDs, which act through calcium mobilization, Gs-DREADDs enhance cellular activity via protein kinase A (PKA)-dependent pathways. These receptors have been used to study neuromodulatory processes, particularly in mood regulation and learning. Research in Molecular Psychiatry (2019) showed that Gs-DREADD activation in the hippocampus enhances long-term memory consolidation. Additionally, increasing cAMP signaling in the prefrontal cortex has been linked to antidepressant-like effects.

Targeted Expression Methods

Precise DREADD receptor expression requires strategies ensuring specificity at the cellular, regional, and temporal levels. Viral vectors, particularly AAVs, are commonly used due to their ability to drive stable, long-term expression with minimal cytotoxicity. Different AAV serotypes exhibit distinct tropisms, allowing researchers to selectively target neuronal subtypes or non-neuronal cells. For instance, AAV5 efficiently transduces dopaminergic neurons, while AAV9 crosses the blood-brain barrier, enabling systemic delivery.

Promoter selection further refines specificity by restricting expression to defined cellular populations. The CaMKIIα promoter drives expression in excitatory pyramidal neurons, while the hSyn promoter provides more generalized neuronal expression. For glial targeting, promoters such as GFAP (astrocytes) or Iba1 (microglia) ensure selective receptor expression. Cre-loxP recombination enables even greater specificity by activating DREADD expression only in genetically defined populations.

Intracellular Signaling Cascades

Once activated, DREADD receptors initiate intracellular signaling cascades that regulate neurotransmitter release, gene expression, and synaptic plasticity. These pathways shape cellular responses based on the specific G protein involved.

Gq-coupled DREADDs activate PLC, producing IP3 and DAG. IP3 mobilizes calcium from intracellular stores, activating kinases and phosphatases that modulate synaptic strength. This pathway plays a critical role in synaptic plasticity, influencing long-term potentiation (LTP) and long-term depression (LTD), mechanisms essential for learning and memory.

Gi-coupled DREADDs suppress adenylyl cyclase activity, reducing cAMP levels and dampening PKA signaling. This inhibition decreases neurotransmitter release and neuronal excitability, making Gi-DREADDs useful in models of epilepsy, addiction, and anxiety disorders.

Gs-coupled DREADDs stimulate adenylyl cyclase, increasing cAMP levels and activating PKA. This cascade enhances synaptic plasticity and promotes gene expression linked to neuronal survival and resilience. Their ability to amplify excitatory signaling has been explored in mood disorders, where dysregulated cAMP signaling is implicated in depression and bipolar disorder.

Commonly Studied Biological Systems

DREADD technology has been widely applied in neuroscience but extends to cardiovascular physiology, immunology, and metabolic regulation. The ability to selectively activate or inhibit specific cell populations has provided insights into organ function and disease mechanisms.

In neuroscience, DREADDs have been used to map circuits underlying cognition, emotion, and motor control. Gi-coupled DREADDs have inhibited dopaminergic neurons in the ventral tegmental area, reducing drug-seeking behavior. Gq-coupled DREADDs have enhanced excitability in the prefrontal cortex, improving cognitive flexibility in schizophrenia models.

Beyond the brain, DREADDs have been used in cardiac research to study heart rate regulation and contractility and in metabolic studies to examine feeding behavior and glucose homeostasis. Their ability to manipulate cellular activity with synthetic ligands has expanded their applications across multiple physiological domains.