Chemogenetic Activation of CX3CR1-Expressing Spinal Microglia Using Gq-DREADD Elicits Mechanical Allodynia in Male Mice

Abstract

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+ 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.

Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

“…Of note, such intracellular calcium increases occur during insults such as epilepsy, focal damage, and lipopolysaccharides (LPS) ( Pozner et al, 2015 ; Umpierre et al, 2020 ). Activating Gq-DREADDs in microglia with CNO also increases markers of microglial activation such as Iba-1 and CD11b and their production of proinflammatory cytokines such as interleukin-1 β (IL-1β) and tumor necrosis factor α (TNF-α) ( Saika et al, 2021 ; Grace et al, 2018 ), further supporting the approach taken here.…”

Microglial inflammatory activation paradoxically improves glucose tolerance during diet-induced obesity

“…To validate Gq-coupled signal transduction in this chemogenetic mouse model, we measured the change in intracellular calcium concentration ([Ca 2+ ] i ) in response to CNO treatment in primary microglia preloaded with the calcium indicator Fura Red. CNO treatment (0.01-0.1 μM) caused a rapid rise in [Ca 2+ ] i , which remained elevated for several hours afterward (Figures 5C and 5D), consistent with prior reports using DREADDs in microglia (Binning et al, 2020; Grace et al, 2018; Saika et al, 2020, 2021; Yi et al, 2021). In vivo , CNO treatment (1 mg/kg; i.p.)…”

“…Though largely applied to studies of neuronal functions, it has also been used to activate a variety of other cell types, both in the CNS and the periphery (Wang et al, 2021). While we and others have used the hM3Dq-DREADD in microglia as a means of cellular stimulation (Binning et al, 2020; Grace et al, 2018; Saika et al, 2020, 2021; Yi et al, 2021), it is a relatively unrefined mechanism of activation and lacks the nuances of environmental adaptation now known to be the cornerstone of microglial function in health and disease (Colonna and Butovsky, 2017; Prinz et al, 2019, 2021; Salter and Stevens, 2017). In this case, use of hM3D mice enabled us to selectively induce microglial activation and cytokine production, recapitulating key aspects of our A20 model and suggesting that microglial inflammatory mediators acutely impact glucose tolerance.…”

Regulation of Neural Circuitry under General Anesthesia: New Methods and Findings

“…CNO binding to hM3Dq excites neurons by increasing intracellular calcium levels, whereas binding to hM4Di silences neuronal activity by reducing adenylate cyclase content [ 66 ]. In addition to the transgenic mouse lines expressing DREADDs [ 67 ], chemogenetic technology requires virus delivery to achieve specific and stable expression of hM3Dq or hM4Di in particular cells in one or more brain regions ( Figure 2 ). Currently, DREADDs are widely applied to regulate specific neural activities and behaviors in many species, such as flies, mice, and even nonhuman primates [ 68 , 69 ].…”