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 G_q- and G_i-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 G_q (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 G_i (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with G_q or G_i DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing G_q-DREADDs, and inhibited lipopolysaccharide- and CCL2-induced inflammatory signaling from cells expressing G_i-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.