Elsevier

Experimental Neurology

Volume 304, June 2018, Pages 125-131
Experimental Neurology

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

https://doi.org/10.1016/j.expneurol.2018.03.005Get rights and content

Highlights

  • DREADDs under a CD68 promoter were intrathecally transfected via an AAV9 vector.

  • Activation of microglia via Gq DREADDs induced allodynia.

  • Inhibition of microglia via Gi DREADDs reversed neuropathic pain.

  • Activation of BV-2 cells via Gq DREADDs induced proinflammatory cytokines.

  • Inhibition of BV-2 cells via Gi DREADDs attenuated LPS- and CCL2-induced cytokines.

Abstract

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.

Introduction

Microglia, the resident macrophages of the central nervous system (CNS), have a key role in maintaining homeostasis and in driving many CNS pathologies, including persistent pain (Grace et al. 2014; Ji et al. 2016; Old et al. 2015; Salter and Stevens 2017). Precise delineation of their function in vivo has been hindered by a lack of tools to selectively activate or inhibit microglia. There are no strategies in routine use to selectively activate microglia without activating other cell types as well. In addition, several approaches have been used to inhibit microglia, but all are flawed with respect to specificity. For example, pharmacological agents such as minocycline, which do inhibit microglia (Yrjänheikki et al. 1998), also have inhibitory effects on other cells in the CNS like neurons, astrocytes and T cells (e.g., (González et al. 2007; Song et al. 2016; Szeto et al. 2011)). Selective depletion methods have been described, such as CSF-1R antagonism (Elmore et al. 2014), or cre-driven expression of the diphtheria toxin receptor in microglia (Parkhurst et al. 2013). However, these methods widely perturb the CNS, as cytokine production and/or astrogliosis is reported. These off-target effects may confound interpretations regarding the role of microglia in physiological and pathological processes.

We have recently reported that hM4Di Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) can be used to inhibit microglia in vitro and in vivo (Grace et al. 2016a). Once expressed under a cell-specific promoter, DREADDs can be remotely activated with clozapine-N-oxide (CNO) (Armbruster et al. 2007). Gi-linked signaling was predicted to attenuate microglial reactivity because activation of the M4 muscarinic receptor (the Gi DREADD progenitor (Armbruster et al. 2007)) inhibits Ca2+ influx in parasympathetic neurons (Cuevas and Adams 1997), a process associated with decreased proinflammatory cytokine production in microglia (Hayashi et al. 2011; Hoffmann et al. 2003). When spinal microglia were transfected with hM4Di DREADDs under a CD68 promoter in vivo, treatment with CNO prevented and reversed morphine-induced persistent sensitization in male rats (Grace et al. 2016a). CNO also attenuated inflammatory gene expression in hM4Di+ BV-2 cells (a microglia cell line) that were stimulated with the danger signal HMGB1 (Grace et al. 2016a; Lacagnina et al. 2017). Inflammatory mediators, such as nitric oxide (NO) and interleukin-1β (IL-1β) promote neuronal hyperexcitability in the spinal dorsal horn (Grace et al., 2014, Grace et al., 2016b). However, it is not known whether these results apply to neuropathic pain per se, and extend to other BV-2 cell stimuli in vitro.

hM3Dq DREADDs have also been developed that mobilize intracellular calcium through Gq proteins (Armbruster et al. 2007). As noted above, increased intracellular Ca2+ is a substrate for microglial activation and cytokine release (Hoffmann et al. 2003). Whether hM3Dq DREADDs can activate microglia is unknown.

The first aim of this study was to determine whether activation or inhibition of microglia via DREADDs could influence nociceptive hypersensitivity. The second aim was to validate behavioral results in microglia cell cultures by determining whether DREADD activation or inhibition would influence cytokine transcription and translation. Our data indicate that DREADDs can be used to modulate microglial activity in vitro and in vivo.

Section snippets

Subjects

Pathogen-free adult male Sprague Dawley rats (n = 6 rats/group for each experiment; 10–12 weeks old on arrival; Envigo Labs, Indianapolis, IN) were used in all experiments. Rats were housed in temperature-controlled (23 ± 3 °C) and light-controlled (12 h light:dark cycle; lights on at 07:00 h) rooms with standard rodent chow and water available ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado Boulder.

DREADDs and drugs

hM4Di inhibition of microglia reverses neuropathic pain

To test whether DREADD inhibition of spinal microglia could reverse established neuropathic pain, rats were transfected with intrathecal CD68-hM4Di or the control vector CD68-eGFP. We have previously validated spinal microglial expression of DREADDs when transfected via an AAV9 vector (Grace et al. 2016a). Two weeks after CCI surgery, a single intrathecal dose of CNO selectively reversed allodynia in those rats expressing hM4Di versus control (Fig. 1a; time x treatment: F4,36 = 34.36, P

Discussion

We found that inhibition of spinal microglia via hM4Di DREADDs attenuated established allodynia and CD11b expression in the dorsal horn of the lumbar spinal cord after peripheral nerve injury. Furthermore, these DREADDs attenuated inflammatory signaling induced by LPS and CCL2 in vitro, complementing similar results with HMGB1 (Grace et al. 2016a). In contrast, activation of spinal microglia via hM3Dq DREADDs induced allodynia in naïve rats, which could be prevented by inhibiting IL-1

Acknowledgements

The authors gratefully acknowledge Dr. Daniel Urban and Dr. Bryan Roth who gifted the DREADDs. This work was supported by the American Pain Society Future Leaders in Pain Research Grants Program (P.M.G.); National Health and Medical Research Council CJ Martin Fellowship ID 1054091 (P.M.G.); American Australian Association Sir Keith Murdoch Fellowship (P.M.G.); National Natural Science Foundation of China Grants 21750110432 (P.M.G.), 21602216, and 21543013 (X.W.); National Key Research and

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  • Cited by (0)

    1

    Current address: Department of Critical Care and Respiratory Care Research, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

    2

    Co-first authors.

    3

    Current address: Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China.

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