biostudies-literatureZhao SU.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and StrokeNIA NIH HHSNIEHS NIH HHSU.S. Department of Health & Human Services | NIH | National Institute of Environmental Health SciencesNINDS NIH HHS2857-2871https://www.ebi.ac.uk/biostudies/studies/S-EPMC10906107biostudies-literatureUnknown28(7)Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1^CreER/+:R26^hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1⁺ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1⁺ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119^CreER/+:R26^hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4⁺ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1^CreER/+ mouse line to manipulate microglia.Molecular psychiatryChemogenetic manipulation of CX3CR1⁺ cells transiently induces hypolocomotion independent of microglia.PMC10906107R35NS132326R35 NS132326R01 ES033892R01ES033892K99 NS126417K99NS126417R01 NS110949RF1 AG082314Zheng JUmpierre ADDheer AZhao SRichardson JRXie MJohnson AJAyasoufi KWang LParusel SWu LJfalseChemogenetic manipulation of CX3CR1⁺ cells transiently induces hypolocomotion independent of microglia.Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1^CreER/+:R26^hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1⁺ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1⁺ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119^CreER/+:R26^hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4⁺ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1^CreER/+ mouse line to manipulate microglia.2023-01-01T00:00:00Z2023 Jul2026-05-29T17:29:35.843Z2026-04-08T05:37:41.685ZS-EPMC109061073736523910.1038/s41380-023-02128-6