Designer Receptor Exclusively Activated By A Designer Drug Research Articles

Acute cannabinoids impair association learning via selectively enhancing synaptic transmission in striatonigral neurons

BackgroundCannabinoids and their derivatives attract strong interest due to the tremendous potential of their psychoactive effects for treating psychiatric disorders and symptoms. However, their clinical application is restricted by various side-effects such as impaired coordination, anxiety, and learning and memory disability. Adverse impact on dorsal striatum-dependent learning is an important side-effect of cannabinoids. As one of the most important forms of learning mediated by the dorsal striatum, reinforcement learning is characterized by an initial association learning phase, followed by habit learning. While the effects of cannabinoids on habit learning have been well-studied, little is known about how cannabinoids influence the initial phase of reinforcement learning.ResultsWe found that acute activation of cannabinoid receptor type 1 (CB1R) by the synthetic cannabinoid HU210 induced dose-dependent impairment of association learning, which could be alleviated by intra-dorsomedial striatum (DMS) injection of CB1R antagonist. Moreover, acute exposure to HU210 elicited enhanced synaptic transmission in striatonigral “direct” pathway medium spiny neurons (MSNs) but not indirect pathway neurons in DMS. Intriguingly, enhancement of synaptic transmission that is also observed after learning was abolished by HU210, indicating cannabinoid system might disrupt reinforcement learning by confounding synaptic plasticity normally required for learning. Remarkably, the impaired response-reinforcer learning was also induced by selectively enhancing the D1-MSN (MSN that selectively expresses the dopamine receptor type 1) activity by virally expressing excitatory hM3Dq DREADD (designer receptor exclusively activated by a designer drug), which could be rescued by specifically silencing the D1-MSN activity via hM4Di DREADD.ConclusionOur findings demonstrate dose-dependent deleterious effects of cannabinoids on association learning by disrupting plasticity change required for learning associated with the striatal direct pathway, which furthers our understanding of the side-effects of cannabinoids and the underlying mechanisms.

Chemogenetic modulation of sensory neurons reveals their regulating role in melanoma progression

Sensory neurons have recently emerged as components of the tumor microenvironment. Nevertheless, whether sensory neuronal activity is important for tumor progression remains unknown. Here we used Designer Receptors Exclusively Activated by a Designer Drug (DREADD) technology to inhibit or activate sensory neurons’ firing within the melanoma tumor. Melanoma growth and angiogenesis were accelerated following inhibition of sensory neurons’ activity and were reduced following overstimulation of these neurons. Sensory neuron-specific overactivation also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of melanoma biopsies revealed that increased expression of sensory neurons-related genes within melanoma was associated with improved survival. These findings suggest that sensory innervations regulate melanoma progression, indicating that manipulation of sensory neurons’ activity may provide a valuable tool to improve melanoma patients’ outcomes.

Chemogenetic approaches to identify metabolically important GPCR signaling pathways: Therapeutic implications.

DREADDs (Designer Receptors Exclusively Activated by a Designer Drug) are designer G protein-coupled receptors (GPCRs) that are widely used in the neuroscience field to modulate neuronal activity. In this review, we will focus on DREADD studies carried out with genetically engineered mice aimed at elucidating signaling pathways important for maintaining proper glucose and energy homeostasis. The availability of muscarinic receptor-based DREADDs endowed with selectivity for one of the four major classes of heterotrimeric G proteins (Gs , Gi , Gq , and G12 ) has been instrumental in dissecting the physiological and pathophysiological roles of distinct G protein signaling pathways in metabolically important cell types. The novel insights gained from this work should inform the development of novel classes of drugs useful for the treatment of several metabolic disorders including type 2 diabetes and obesity.

Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs.

G protein-coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.

1781-P: Adipocyte GI Signaling Regulates Whole-Body Glucose Homeostasis and Insulin Sensitivity

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes (T2D). Receptor-mediated activation of heterotrimeric G proteins plays a pivotal role in regulating adipocyte function. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this issue, we generated adipocre-ROSA 26PTXmice in which Gisignaling was selectively disrupted in adipocytes ('adipo-Gi KO mice'). Compared to control mice, adipo-Gi KO mice displayed increased lipolysis and decreased fat mass when maintained on regular chow or a high fat diet (HFD). Moreover, HFD adipo-Gi KO mice showed impaired glucose tolerance and reduced insulin sensitivity. In vitro studies with primary adipocytes prepared from adipo-Gi KO mice indicated that the lack of Gisignaling led to impaired insulin signaling and reduced insulin-stimulated glucose uptake. In parallel, we also used a chemo-genetic approach to generate a mouse line selectively expressing a CNO-sensitive, Gi-coupled DREADD (designer receptor exclusively activated by a designer drug) in adipocytes (adipo-GiD mice). CNO-induced activation of adipocyte Gi signaling in adipo-GiD mice significantly improved glucose tolerance and insulin sensitivity independent of the diet that the mice consumed (regular chow or HFD). In vivo 14C-2-deoxy-glucose uptake assays demonstrated that activation of adipocyte Gisignaling enhanced insulin-induced glucose uptake in adipose tissues. Stimulation of adipocyte Gisignaling activation also further enhanced insulin signaling in adipose tissues. Taken together, our data clearly demonstrate that adipocyte Gisignaling is a critical regulator of insulin signaling in adipocytes and is critical for the maintenance of glucose homeostasis. A better understanding of the mechanisms through which Gipromotes adipocyte insulin signaling should stimulate the development of novel antidiabetic drugs. Disclosure L. Wang: None. S. Pydi: None. L. Zhu: None. S. Jain: None. Y. Cui: None. O. Gavrilova: None. J. Wess: None. Funding National Institutes of Health

44-OR: Human Pseudo Islets Reveal Differential Effects of Gq Signaling in ß Cells in Low and High Glucose

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p<0.05 β and α cells). In perifusion, pseudoislets were similar to native islets in biphasic glucose-stimulated and cAMP-potentiated insulin secretion as well as in epinephrine and low glucose stimulation of and high glucose inhibition of glucagon release. Adenoviral-transduced dispersed human islet cells (>90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p<0.0001). In contrast, hM3Dq and control pseudoislets had similar insulin secretion in high glucose (11mM G + 10μM CNO; 24.8 vs. 23.2 ng/100IEQ/hr; p>0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.

Pharmacogenetic neuronal stimulation increases human tau pathology and trans-synaptic spread of tau to distal brain regions in mice

In Alzheimer's Disease (AD), tau pathology has a spatiotemporally distinct pattern of progressive spread along anatomically connected neural pathways. Extracellular tau in the brain interstitial space increases in response to neuronal activity suggesting that neural activity may also drive pathogenic tau spread. Here we tested the hypothesis that neuronal activity drives human Tau (hTau) release and trans-synaptic spread to neuroanatomically connected regions. We used AAV to overexpress wild type full-length hTau and an excitatory DREADD (Designer Receptors Exclusively Activated by a Designer Drug) in mouse primary hippocampal cultures and determined that excitatory stimulation with the DREADD ligand clozapine N-oxide (CNO) promoted extracellular hTau release. We translated this approach to an in vivo model and used AAV to express hTau and the excitatory DREADD in the ventral hippocampus of wild type mice, P301L hTau-expressing mice, or tau knockout mice. Six to eight weeks following AAV injection, we determined that CNO treatment in DREADD-expressing mice resulted in increased hTau pathology and hTau spread to distal brain regions compared to unstimulated controls (CNO in non-DREADD mice, or vehicle in DREADD mice). The results highlight a potentially disease relevant exacerbation of tau pathology in response to elevated neuronal activity. This model underscores the propensity of non-mutant hTau to undergo neuronal spreading, as seen in AD. The model can translate to other preclinical species and can be used to evaluate modes of tau transmission and test the efficacy of therapeutic approaches that target tau or hyperexcitability.

Activation of catecholamine neurons in the ventral medulla reduces CCK-induced hypophagia and c-Fos activation in dorsal medullary catecholamine neurons.

Catecholamine (CA) neurons within the A1 and C1 cell groups in the ventrolateral medulla (VLM) potently increase food intake when activated by glucose deficit. In contrast, CA neurons in the A2 cell group of the dorsomedial medulla are required for reduction of food intake by cholecystokinin (CCK), a peptide that promotes satiation. Thus dorsal and ventral medullary CA neurons are activated by divergent metabolic conditions and mediate opposing behavioral responses. Acute glucose deficit is a life-threatening condition, and increased feeding is a key response that facilitates survival of this emergency. Thus, during glucose deficit, responses to satiation signals, like CCK, must be suppressed to ensure glucorestoration. Here we test the hypothesis that activation of VLM CA neurons inhibits dorsomedial CA neurons that participate in satiation. We found that glucose deficit produced by the antiglycolytic glucose analog, 2-deoxy-d-glucose, attenuated reduction of food intake by CCK. Moreover, glucose deficit increased c-Fos expression by A1 and C1 neurons while reducing CCK-induced c-Fos expression in A2 neurons. We also selectively activated A1/C1 neurons in TH-Cre+ transgenic rats in which A1/C1 neurons were transfected with a Cre-dependent designer receptor exclusively activated by a designer drug (DREADD). Selective activation of A1/C1 neurons using the DREADD agonist, clozapine- N-oxide, attenuated reduction of food intake by CCK and prevented CCK-induced c-Fos expression in A2 CA neurons, even under normoglycemic conditions. Results support the hypothesis that activation of ventral CA neurons attenuates satiety by inhibiting dorsal medullary A2 CA neurons. This mechanism may ensure that satiation does not terminate feeding before restoration of normoglycemia.

Activation of parvalbumin interneurons in anterior cingulate cortex impairs observational fear

The ability to detect conspecific’s distress is crucial for animal survival. In rodent models, observational fear (OF) occurs when one animal perceives another fear related negative emotions, which may model certain behaviors caused by witnessing traumatic experiences in humans. Anterior cingulate cortex (ACC) has been showed to play a crucial role in OF. However, cellular and neural circuit basis relating to ACC governing OF is poorly understood. Here, we used Designer Receptor Exclusively Activated by a Designer Drug (DREADD) system to investigate the cell type specific circuit mechanism of ACC in OF. Firstly, inhibitory hM4D (Gi) designer receptor together with clozapine N-oxide (CNO) injection was applied to inactivate ACC neurons in the observer mice. We found that, chemogenetic inhibition of ACC resulted in a decreased freezing response in the observer mice. Next, combining PV-ires-Cre mice and Cre-dependent DREADD system, we selectively targeted the ACC parvalbumin (PV) interneurons with the excitatory hM3D (Gq) designer receptor. Activation of ACC PV interneurons following CNO injection reduced freezing response in the observer mice, while had no effect on freezing response in the demonstrator mice. Finally, monosynaptic rabies retrograde tracing revealed that ACC PV interneurons receive inputs from the mediodorsal thalamic nucleus (MD) and the ventromedial thalamic nucleus (VM), both known for their roles in OF. Taken together, these findings reveal that ACC activation is important for OF, during which PV interneurons in ACC play an important regulatory role. Abnormal function of ACC PV interneurons might contribute to the pathology of empathy- deficits related diseases, such as autism and schizophrenia.

Gi protein functions in thalamic neurons to decrease orofacial nociceptive response

Orofacial pain includes neuronal pathways that project from the trigeminal nucleus to and through the thalamus. What role the ventroposterior thalamic complex (VP) has on orofacial pain transmission is not understood. To begin to address this question an inhibitory G protein (Gi) designer receptor exclusively activated by a designer drug (DREADD) was transfected in cells of the VP using adeno-associated virus isotype 8. Virus infected cells were identified by a fluorescent tag and immunostaining. Cells were silenced after injecting the designer drug clozapine-n-oxide, which binds the designer receptor activating Gi. Facial rubbing and local field potentials (LFP) in the VP were then recorded in awake, free moving Sprague Dawley rats after formalin injection of the masseter muscle to induce nociception. Formalin injection significantly increased LFP and the nociceptive behavioral response. Activation of DREADD Gi with clozapine-n-oxide significantly reduced LFP in the VP and reduced the orofacial nociceptive response. Because DREADD silencing can result from Gi-coupled inwardly-rectifying potassium channels (GIRK), the GIRK channel blocker tertiapin-Q was injected. Injection of GIRK blocker resulted in an increase in the nociceptive response and increased LFP activity. Immunostaining of the VP for glutamate vesicular transporter (VGLUT2) and gamma-aminobutyric acid vesicular transporter (VGAT) indicated a majority of the virally transfected cells were excitatory (VGLUT2 positive) and a minority were inhibitory (VGAT positive). We conclude first, that inhibition of the excitatory neurons within the VP reduced electrical activity and the orofacial nociceptive response and that the effect on excitatory neurons overwhelmed any change resulting from inhibitor neurons. Second, inhibition of LFP and nociception was due, in part, to GIRK activation.

DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats

The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a Gq- and Gi-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naïve male rats intrathecally transfected with Gq (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO), which was abolished by intrathecal interleukin-1 receptor antagonist. Chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in male rats intrathecally transfected with Gi (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with Gq or Gi DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing Gq-DREADDs, and inhibited lipopolysaccharide- and CCL2-induced inflammatory signaling from cells expressing Gi-DREADDs. These studies are the first to manipulate microglia function using DREADDs, which allow the role of glia in pain to be conclusively demonstrated, unconfounded by neuronal off-target effects that exist for all other drugs that also inhibit glia. Hence, these studies are the first to conclusively demonstrate that in vivo stimulation of resident spinal microglia in intact spinal cord is a) sufficient for allodynia, and b) necessary for allodynia induced by peripheral nerve injury. DREADDs are a unique tool to selectively explore the physiological and pathological role of microglia in vivo.

Role for the Ventral Posterior Medial/Posterior Lateral Thalamus and Anterior Cingulate Cortex in Affective/Motivation Pain Induced by Varicella Zoster Virus.

Varicella zoster virus (VZV) infects the face and can result in chronic, debilitating pain. The mechanism for this pain is unknown and current treatment is often not effective, thus investigations into the pain pathway become vital. Pain itself is multidimensional, consisting of sensory and affective experiences. One of the primary brain substrates for transmitting sensory signals in the face is the ventral posterior medial/posterior lateral thalamus (VPM/VPL). In addition, the anterior cingulate cortex (ACC) has been shown to be vital in the affective experience of pain, so investigating both of these areas in freely behaving animals was completed to address the role of the brain in VZV-induced pain. Our lab has developed a place escape avoidance paradigm (PEAP) to measure VZV-induced affective pain in the orofacial region of the rat. Using this assay as a measure of the affective pain experience a significant response was observed after VZV injection into the whisker pad and after VZV infusion into the trigeminal ganglion. Local field potentials (LFPs) are the summed electrical current from a group of neurons. LFP in both the VPM/VPL and ACC was attenuated in VZV injected rats after inhibition of neuronal activity. This inhibition of VPM/VPL neurons was accomplished using a designer receptor exclusively activated by a designer drug (DREADD). Immunostaining showed that cells within the VPM/VPL expressed thalamic glutamatergic vesicle transporter-2, NeuN and DREADD suggesting inhibition occurred primarily in excitable neurons. From these results we conclude: (1) that VZV associated pain does not involve a mechanism exclusive to the peripheral nerve terminals, and (2) can be controlled, in part, by excitatory neurons within the VPM/VPL that potentially modulate the affective experience by altering activity in the ACC.

Chemogenetic Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis.

To determine the ability of drugs that activate inhibitory G protein-coupled receptors (GPCRs) expressed in peripheral voltage-gated sodium channel 1.8 (NaV 1.8)-positive sensory neurons to control osteoarthritis (OA)-associated pain. We used designer receptors exclusively activated by a designer drug (DREADD) technology, which employs engineered GPCRs to activate or inhibit neurons upon binding the synthetic ligand clozapine N-oxide (CNO). NaV 1.8-Pdi C57BL/6 mice were generated to express the inhibitory DREADD receptor Pdi in NaV 1.8-expressing sensory neurons. Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male mice. Four, 8, 12, or 16 weeks after surgery, knee hyperalgesia or hind paw mechanical allodynia was tested. Subsequently, CNO or vehicle was administered, and the effect on pain-related behaviors was measured by a blinded observer. Morphine was used as a control. Immunohistochemistry and electrophysiology confirmed functional expression of the inhibitory DREADD receptor Pdi by NaV 1.8-positive sensory neurons. Acute inhibition of NaV 1.8-expressing neurons in mice treated with CNO reduced knee hyperalgesia 4 weeks after DMM surgery and reduced mechanical allodynia 8 weeks after DMM surgery. Inhibition had no effect on pain-related behaviors 12 and 16 weeks after DMM surgery. Morphine, a drug that activates GPCRs in the peripheral and central nervous systems, was still effective in the later stage of experimental OA. Chemogenetic inhibition of NaV 1.8-expressing neurons blocks knee hyperalgesia and mechanical allodynia in early experimental OA, but is no longer efficacious in the later stages. These data indicate that activation of inhibitory GPCRs located solely outside the central nervous system may be ineffective in treating chronic OA pain.

Transient Cell-intrinsic Activity Regulates the Migration and Laminar Positioning of Cortical Projection Neurons.

Neocortical microcircuits are built during development and require the coordinated assembly of excitatory glutamatergic projection neurons (PNs) into functional networks. Neuronal migration is an essential step in this process. In addition to cell-intrinsic mechanisms, external cues including neurotransmitters regulate cortical neuron migration, suggesting that early activity could influence this process. Here, we aimed to investigate the role of cell-intrinsic activity in migrating PNs in vivo using a designer receptor exclusively activated by a designer drug (DREADD) chemogenetic approach. In utero electroporation was used to specifically express the human M3 muscarinic cholinergic Gq-coupled receptor (hM3Dq) in PNs and calcium activity, migratory dynamics, gene expression, and laminar positioning of PNs were assessed following embryonic DREADD activation. We found that transient embryonic DREADD activation induced premature branching and transcriptional changes in migrating PNs leading to a persistent laminar mispositioning of superficial layer PNs into deep cortical layers without affecting expression of layer-specific molecular identity markers. In addition, live imaging approaches indicated that embryonic DREADD activation increased calcium transients in migrating PNs and altered their migratory dynamics by increasing their pausing time. Taken together, these results support the idea that increased cell-intrinsic activity during migration acts as a stop signal for migrating cortical PNs.

Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptors (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-Aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds Designer Receptors Exclusively Activated by a Designer Drug (DREADDs), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression (LTD)-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping (DREAMM) to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area (VTA), and hindbrain structures such as the locus coeruleus (LC) and parabrachial nucleus (PB). These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior, and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Single and Transient Ca2+ Peaks in Podocytes do not induce Changes in Glomerular Filtration and Perfusion

Chronic alterations in calcium (Ca2+) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.

A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways

Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.

Selective manipulation of spinal microglia by chemogenetics: Implications for allodynia and inflammatory signaling

The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a Gq- and Gi-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naive rats intrathecally transfected with Gq (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO), which was abolished by intrathecal IL-1ra. Chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in rats intrathecally transfected with Gi (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with Gq or Gi DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing Gq-DREADDs, and inhibited lipopolysaccharide-induced pro-inflammatory mediator production from cells expressing Gi-DREADDs. These studies are the first to manipulate microglia using DREADDs, which allow the role of glia in pain to be conclusively demonstrated, unconfounded by neuronal off-target effects that exist for all other glial inhibitors. Hence, these studies are the first to conclusively demonstrate that in vivo stimulation of resident spinal microglia in intact spinal cord is (a) sufficient for allodynia, and (b) necessary for allodynia induced by peripheral nerve injury. DREADDs are a unique tool to selectively explore the physiological and pathological role of microglia in vivo.

Chemogenetic Inactivation of Dorsal Anterior Cingulate Cortex Neurons Disrupts Attentional Behavior in Mouse.

Attention is disrupted commonly in psychiatric disorders, yet mechanistic insight remains limited. Deficits in this function are associated with dorsal anterior cingulate cortex (dACC) excitotoxic lesions and pharmacological disinhibition; however, a causal relationship has not been established at the cellular level. Moreover, this association has not yet been examined in a genetically tractable species such as mice. Here, we reveal that dACC neurons causally contribute to attention processing by combining a chemogenetic approach that reversibly suppresses neural activity with a translational, touchscreen-based attention task in mice. We virally expressed inhibitory hM4Di DREADD (designer receptor exclusively activated by a designer drug) in dACC neurons, and examined the effects of this inhibitory action with the attention-based five-choice serial reaction time task. DREADD inactivation of the dACC neurons during the task significantly increased omission and correct response latencies, indicating that the neuronal activities of dACC contribute to attention and processing speed. Selective inactivation of excitatory neurons in the dACC not only increased omission, but also decreased accuracy. The effect of inactivating dACC neurons was selective to attention as response control, motivation, and locomotion remain normal. This finding suggests that dACC excitatory neurons play a principal role in modulating attention to task-relevant stimuli. This study establishes a foundation to chemogenetically dissect specific cell-type and circuit mechanisms underlying attentional behaviors in a genetically tractable species.

Gq-DREADD Selectively Initiates Glial Glutamate Release and Inhibits Cue-induced Cocaine Seeking

BackgroundGlial cells of the central nervous system directly influence neuronal activity by releasing neuroactive small molecules, including glutamate. Long-lasting cocaine-induced reductions in extracellular glutamate in the nucleus accumbens core (NAcore) affect synaptic plasticity responsible for relapse vulnerability. MethodsWe transduced NAcore astrocytes with an adeno-associated virus vector expressing hM3D designer receptor exclusively activated by a designer drug (DREADD) under control of the glial fibrillary acidic protein promoter in 62 male Sprague Dawley rats, 4 dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor mice, and 4 wild-type littermates. Using glutamate biosensors, we measured NAcore glutamate levels following intracranial or systemic administration of clozapine N-oxide (CNO) and tested the ability of systemic CNO to inhibit reinstated cocaine or sucrose seeking following self-administration and extinction training. ResultsAdministration of CNO in glial fibrillary acidic protein-hM3D-DREADD transfected animals increased NAcore extracellular glutamate levels in vivo. The glial origin of released glutamate was validated by an absence of CNO-mediated release in mice expressing a dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor variant in glia. Also, CNO-mediated release was relatively insensitive to N-type calcium channel blockade. Systemic administration of CNO inhibited cue-induced reinstatement of cocaine seeking in rats extinguished from cocaine but not sucrose self-administration. The capacity to inhibit reinstated cocaine seeking was prevented by systemic administration of the group II metabotropic glutamate receptor antagonist LY341495. ConclusionsDREADD-mediated glutamate gliotransmission inhibited cue-induced reinstatement of cocaine seeking by stimulating release-regulating group II metabotropic glutamate receptor autoreceptors to inhibit cue-induced synaptic glutamate spillover.