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(250) 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, prior to any experimental manipulation. This was done so to ensure minimal transfection of macrophages relative to transfection of resident microglia. Naïve rats intrathecally transfected with Gq (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO). This microglia-specific allodynia was abolished by intrathecal interleukin-1 receptor antagonist, demonstrating mediation of allodynia by interleukin-1. In contrast, chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in rats intrathecally transfected with Gi (inhibitory) DREADDs. To explore the mechanisms of DREADD modification of glial reactivity, BV2 cells were stably transfected with Gi (inhibitory) DREADDs in vitro, where pro-inflammatory mediator production, induced by lipopolysaccharide, was attenuated following CNO treatment. 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 known to 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.