Chemogenetic Regulation of CX3CR1-Expressing Microglia Using Gi-DREADD Exerts Sex-Dependent Anti-Allodynic Effects in Mouse Models of Neuropathic Pain.
ABSTRACT: Despite growing evidence suggesting that spinal microglia play an important role in the molecular mechanism underlying experimental neuropathic pain (NP) in male rodents, evidence regarding the sex-dependent role of these microglia in NP is insufficient. In this study, we evaluated the effects of microglial regulation on NP using Gi-designer receptors exclusively activated by designer drugs (Gi-DREADD) driven by the microglia-specific Cx3cr1 promoter. For the Cre-dependent expression of human Gi-coupled M4 muscarinic receptors (hM4Di) in CX3C chemokine receptor 1-expressing (CX3CR1+) cells, R26-LSL-hM4Di-DREADD mice were crossed with CX3CR1-Cre mice. Mouse models of NP were generated by partial sciatic nerve ligation (PSL) and treatment with anti-cancer agent paclitaxel (PTX) or oxaliplatin (OXA), and mechanical allodynia was evaluated using the von Frey test. Immunohistochemistry revealed that hM4Di was specifically expressed on Iba1+ microglia, but not on astrocytes or neurons in the spinal dorsal horn of CX3CR1-hM4Di mice. PSL-induced mechanical allodynia was significantly attenuated by systemic (intraperitoneal, i.p.) administration of 10 mg/kg of clozapine N-oxide (CNO), a hM4Di-selective ligand, in male CX3CR1-hM4Di mice. The mechanical threshold in naive CX3CR1-hM4Di mice was not altered by i.p. administration of CNO. Consistently, local (intrathecal, i.t.) administration of CNO (20 nmol) significantly relieved PSL-induced mechanical allodynia in male CX3CR1-hM4Di mice. However, neither i.p. nor i.t. administration of CNO affected PSL-induced mechanical allodynia in female CX3CR1-hM4Di mice. Both i.p. and i.t. administration of CNO relieved PTX-induced mechanical allodynia in male CX3CR1-hM4Di mice, and a limited effect of i.p. CNO was observed in female CX3CR1-hM4Di mice. Unlike PTX-induced allodynia, OXA-induced mechanical allodynia was slightly improved, but not significantly relieved, by i.p. administration of CNO in both male and female CX3CR1-hM4Di mice. These results suggest that spinal microglia can be regulated by Gi-DREADD and support the notion that CX3CR1+ spinal microglia play sex-dependent roles in nerve injury-induced NP; however, their roles may vary among different models of NP.
Project description:It is important to investigate the sex-dependent roles of microglia in pain hypersensitivity as reactive microglia within the spinal dorsal horn (DH) have been reported to be pivotal in neuropathic pain induction in male rodents upon nerve injury. Here, we aimed at determining the role of sex differences in the behavioral and functional outcomes of the chemogenetic activation of spinal microglia using Gq-designer receptors exclusively activated by designer drugs (Gq-DREADD) driven by the microglia-specific Cx3cr1 promoter. CAG-LSL-human Gq-coupled M3 muscarinic receptors (hM3Dq)-DREADD mice were crossed with CX3C chemokine receptor 1 (CX3CR1)-Cre mice, and immunohistochemistry images revealed that hM3Dq was selectively expressed on Iba1<sup>+</sup> microglia, but not on astrocytes and neurons. Intrathecal (i.t.) administration of clozapine-N-oxide (CNO) elicited mechanical allodynia exclusively in male mice. Furthermore, the reactive microglia-dominant molecules that contributed to pain hypersensitivity in CX3CR1-hM3Dq were upregulated in mice of both sexes. The degree of upregulation was greater in male than in female mice. Depletion of spinal microglia using pexidartinib (PLX3397), a colony stimulating factor-1 receptor inhibitor, alleviated the male CX3CR1-hM3Dq mice from pain hypersensitivity and compromised the expression of inflammatory molecules. Thus, the chemogenetic activation of spinal microglia resulted in pain hypersensitivity in male mice, suggesting the sex-dependent molecular aspects of spinal microglia in the regulation of pain.
Project description:<h4>Background</h4>Neuro-immune interaction underlies chronic neuroinflammation and aberrant sensory processing resulting in neuropathic pain. Despite the pathological significance of both neuroinflammation-driven peripheral sensitization and spinal sensitization, the functional relationship between these two distinct events has not been understood.<h4>Methods</h4>In this study, we determined whether inhibition of inflammatory macrophages by administration of ?4?2 nicotinic acetylcholine receptor (nAChR) agonists improves neuropathic pain and affects microglial activation in the spinal dorsal horn (SDH) in mice following partial sciatic nerve ligation (PSL). Expression levels of neuroinflammatory molecules were evaluated by RT-qPCR and immunohistochemistry, and PSL-induced mechanical allodynia was defined by the von Frey test.<h4>Results</h4>Flow cytometry revealed that CD11b<sup>+</sup> F4/80<sup>+</sup> macrophages were accumulated in the injured sciatic nerve (SCN) after PSL. TC-2559, a full agonist for ?4?2 nAChR, suppressed the upregulation of interleukin-1? (IL-1?) in the injured SCN after PSL and attenuated lipopolysaccharide-induced upregulation of IL-1? in cultured macrophages. Systemic (subcutaneous, s.c.) administration of TC-2559 during either the early (days 0-3) or middle/late (days 7-10) phase of PSL improved mechanical allodynia. Moreover, local (perineural, p.n.) administration of TC-2559 and sazetidine A, a partial agonist for ?4?2 nAChR, during either the early or middle phase of PSL improved mechanical allodynia. However, p.n. administration of sazetidine A during the late (days 21-24) phase did not show the attenuating effect, whereas p.n. administration of TC-2559 during this phase relieved mechanical allodynia. Most importantly, p.n. administration of TC-2559 significantly suppressed morphological activation of Iba1<sup>+</sup> microglia and decreased the upregulation of inflammatory microglia-dominant molecules, such as CD68, interferon regulatory factor 5, and IL-1? in the SDH after PSL.<h4>Conclusion</h4>These findings support the notion that pharmacological inhibition of inflammatory macrophages using an ?4?2 nAChR agonist exhibit a wide therapeutic window on neuropathic pain after nerve injury, and it could be nominated as a novel pharmacotherapy to relieve intractable pain.
Project description:<h4>Background and purpose</h4>Ischemic stroke significantly perturbs neuronal homeostasis leading to a cascade of pathologic events causing brain damage. In this study, we assessed acute stroke outcome after chemogenetic inhibition of forebrain excitatory neuronal activity.<h4>Methods</h4>We generated hM4Di-TG transgenic mice expressing the inhibitory hM4Di, a Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic receptor, in forebrain excitatory neurons. Clozapine-N-oxide (CNO) was used to activate hM4Di DREADD. Ischemic stroke was induced by transient occlusion of the middle cerebral artery. Neurologic function and infarct volumes were evaluated. Excitatory neuronal suppression in the hM4Di-TG mouse forebrain was assessed electrophysiologically in vitro and in vivo, based on evoked synaptic responses, and in vivo based on occurrence of potassium-induced cortical spreading depolarizations.<h4>Results</h4>Detailed characterization of hM4Di-TG mice confirmed that evoked synaptic responses in both in vitro hippocampal slices and in vivo motor cortex were significantly reduced after CNO-mediated activation of the inhibitory hM4Di DREADD. Further, CNO treatment had no obvious effects on physiology and motor function in either control or hM4Di-TG mice. Importantly, hM4Di-TG mice treated with CNO at either 10 min before ischemia or 30 min after reperfusion exhibited significantly improved neurologic function and smaller infarct volumes compared to CNO-treated control mice. Mechanistically, we showed that potassium-induced cortical spreading depression episodes were inhibited, including frequency and duration of DC shift, in CNO-treated hM4Di-TG mice.<h4>Conclusions</h4>Our data demonstrate that acute inhibition of a subset of excitatory neurons after ischemic stroke can prevent brain injury and improve functional outcome. This study, together with the previous work in optogenetic neuronal modulation during the chronic phase of stroke, supports the notion that targeting neuronal activity is a promising strategy in stroke therapy.
Project description:Abstinent alcohol-dependent individuals experience an enduring sensitivity to cue-induced craving and relapse to drinking. There is considerable evidence indicating that structures within the midbrain and extended amygdala are involved in this process. Individually, the ventral tegmental area (VTA) and the bed nucleus of the stria terminalis (BNST) have been shown to modulate cue-induced ethanol-seeking behavior. It is hypothesized that cue-induced seeking is communicated through a direct projection from the BNST to VTA. In the current experiments, an intersectional viral strategy was used in DBA/2J mice to selectively target and inhibit BNST projections to the VTA during a test of ethanol conditioned place preference (CPP). Inhibitory designer receptors exclusively activated by designer drugs (hM4Di DREADDs) were expressed in VTA-projecting BNST (BNST-VTA) cells by infusing a retrograde herpes-simplex virus encoding cre recombinase (HSV-Cre) into VTA and a cre-inducible adeno-associated virus encoding hM4Di (AAV-DIO-hM4Di) into BNST. Before testing the expression of preference, clozapine-N-oxide (CNO) was peripherally administered to activate hM4Di receptors and selectively inhibit these cells. Ethanol CPP expression was blocked by CNO-mediated inhibition of BNST-VTA cells. A follow-up study revealed this effect was specific to CNO activation of hM4Di as saline- and CNO-treated mice infused with a control vector (HSV-GFP) in place of HSV-Cre showed significant CPP. These findings establish a role for a direct BNST input to VTA in cue-induced ethanol-seeking behavior.
Project description: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.
Project description:Microgliosis, the activation of microglial cells, is thought to contribute to synaptic transmission in the dorsal horn and thereby promote chronic pain. The primary aim of this study was to document the temporal profile of dorsal horn microgliosis after destabilization of the medial meniscus (DMM) in wild type (WT) and Adamts5 null mice. Since neuronal fractalkine (CX3CL1) contributes to microgliosis, we assessed its release from dorsal root ganglia (DRG) cultures after DMM.DMM or sham surgery was performed in the right knee of 10-week old male WT, CX3CR1-green fluorescent protein (GFP), or Adamts5 null C57BL/6 mice. Hind paw mechanical allodynia was monitored using von Frey fibers. L4 dorsal horn microgliosis was assessed 4, 8 and 16 weeks after surgery, based on the morphology of Iba1-immunoreactive microglia. DRG cells (L3-L5) were cultured and supernatants collected for fractalkine (FKN) ELISA.In WT mice, numbers of activated microglia were increased 8 and 16 weeks, but not 4 weeks, after DMM but not sham surgery. DRG cultures showed increased basal FKN release at 8 and 16 weeks. Adamts5 null mice did not develop mechanical allodynia up to 16 weeks after DMM. Accordingly, DRG cultures from these mice did not exhibit increased FKN release and dorsal horn microgliosis did not occur.DMM surgery leads to late stage dorsal horn microgliosis. The temporal correlation with DRG FKN release suggests it may contribute to microgliosis. Reduced microgliosis in Adamts5 null mice, which are protected from joint damage and associated mechanical allodynia after DMM, suggests that microgliosis is associated with joint damage and accompanying persistent pain.
Project description:A dose-limiting side effect of chemotherapeutic agents such as vincristine (VCR) is neuropathic pain, which is poorly managed at present. Chemokine-mediated immune cell/neuron communication in preclinical VCR-induced pain forms an intriguing basis for the development of analgesics. In a murine VCR model, CX3CR1 receptor-mediated signalling in monocytes/macrophages in the sciatic nerve orchestrates the development of mechanical hypersensitivity (allodynia). CX3CR1-deficient mice however still develop allodynia, albeit delayed; thus, additional underlying mechanisms emerge as VCR accumulates. Whilst both patrolling and inflammatory monocytes express CX3CR1, only inflammatory monocytes express CCR2 receptors. We therefore assessed the role of CCR2 in monocytes in later stages of VCR-induced allodynia.Mechanically evoked hypersensitivity was assessed in VCR-treated CCR2- or CX3CR1-deficient mice. In CX3CR1-deficient mice, the CCR2 antagonist, RS-102895, was also administered. Immunohistochemistry and Western blot analysis were employed to determine monocyte/macrophage infiltration into the sciatic nerve as well as neuronal activation in lumbar DRG, whilst flow cytometry was used to characterise monocytes in CX3CR1-deficient mice. In addition, THP-1 cells were used to assess CX3CR1-CCR2 receptor interactions in vitro, with Western blot analysis and ELISA being used to assess expression of CCR2 and proinflammatory cytokines.We show that CCR2 signalling plays a mechanistic role in allodynia that develops in CX3CR1-deficient mice with increasing VCR exposure. Indeed, the CCR2 antagonist, RS-102895, proves ineffective in mice possessing functional CX3CR1 receptors but reduces VCR-induced allodynia in CX3CR1-deficient mice, in which CCR2+ monocytes are elevated by VCR. We suggest that a novel interaction between CX3CR1 and CCR2 receptors in monocytes accounts for the therapeutic effect of RS-102895 in CX3CR1-deficient mice. Indeed, we observe that CCR2, along with its ligand, CCL2, is elevated in the sciatic nerve in CX3CR1-deficient mice, whilst in THP-1 cells (human monocytes), downregulating CX3CR1 upregulates CCR2 expression via p38 MAP kinase signalling. We also show that the CX3CR1-CCR2 interaction in vitro regulates the release of pronociceptive cytokines TNF-? and IL1?.Our data suggests that CCL2/CCR2 signalling plays a crucial role in VCR-induced allodynia in CX3CR1-deficient mice, which arises as a result of an interaction between CX3CR1 and CCR2 in monocytes.
Project description:OBJECTIVE: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. METHODS: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. RESULTS: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. CONCLUSION: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.
Project description:Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-<i>N</i>-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5<i>H</i>-dibenzo[<i>b</i>,<i>e</i>][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di <i>in vivo</i> can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for <i>in vivo</i> studies where metabolic conversion of CNO to clozapine is a concern.
Project description:Pain is the most severe and common symptom of endometriosis. Its underlying pathogenetic mechanism is poorly understood. Nerve sensitization is a particular research challenge, due to the limitations of general endometriosis models and sampling nerve tissue from patients. The chemokine fractalkine (FKN) has been demonstrated to play a key role in various forms of neuropathic pain, while its role in endometriotic pain is unknown. Our study was designed to explore the function of FKN in the development and maintenance of peripheral hyperalgesia and central sensitization in endometriosis using a novel endometriosis animal model developed in our laboratory. After modeling, behavioral tests were carried out and the optimal time for molecular changes was obtained. We extracted ectopic tissues and L4-6 spinal cords to detect peripheral and central roles for FKN, respectively. To assess morphologic characteristics of endometriosis-like lesions-as well as expression and location of FKN/CX3CR1-we performed H&E staining, immunostaining, and western blotting analyses. Furthermore, inhibition of FKN expression in the spinal cord was achieved by intrathecal administration of an FKN-neutralizing antibody to demonstrate its function. Our results showed that implanted autologous uterine tissue around the sciatic nerve induced endometriosis-like lesions and produced mechanical hyperalgesia and allodynia. FKN was highly expressed on macrophages, whereas its receptor CX3CR1 was overexpressed in the myelin sheath of sciatic nerve fibers. Overexpressed FKN was also observed in neurons. CX3CR1/pp38-MAPK was upregulated in activated microglia in the spinal dorsal horn. Intrathecal administration of FKN-neutralizing antibody not only reversed the established mechanical hyperalgesia and allodynia, but also inhibited the expression of CX3CR1/pp38-MAPK in activated microglia, which was essential for the persistence of central sensitization. We concluded that the FKN/CX3CR1 signaling pathway might be one of the mechanisms of peripheral hyperalgesia in endometriosis, which requires further studies. Spinal FKN is important for the development and maintenance of central sensitization in endometriosis, and it may further serve as a novel therapeutic target to relieve persistent pain associated with endometriosis.