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. 2023 Jun 25:25:100553.
doi: 10.1016/j.ynstr.2023.100553. eCollection 2023 Jul.

Dysfunctional synaptic pruning by microglia correlates with cognitive impairment in sleep-deprived mice: Involvement of CX3CR1 signaling

Lu Wang  1   2 , Hanyi Ling  2 , Hui He  2 , Nan Hu  2 , Lin Xiao  1 , Yue Zhang  2 , Lei Xie  2 , Zili You  1   2
Affiliations

Dysfunctional synaptic pruning by microglia correlates with cognitive impairment in sleep-deprived mice: Involvement of CX3CR1 signaling

Lu Wang et al. Neurobiol Stress. .

Abstract

Microglia are involved in sleep/wake cycles and the response to sleep loss. Synaptic pruning by microglia is necessary for central nervous system circuit refinement and contributes to cognitive function. Here, we investigated whether and how microglia-mediated synaptic pruning may be involved in cognitive deficits induced by sleep deprivation in mice. Mice were deprived of sleep by leaving them in a spontaneously rotating rod for 72 h, after which their cognitive function was assessed using an object location test, Y maze, and novel object recognition test. Sleep deprivation lowered the discrimination index for familiar locations in the object location test and Y maze. Microglial morphology was assessed using immunostaining Iba1, while microglia-mediated synaptic pruning was examined based on immunostaining PSD95, CD68, and Iba1. Sleep deprivation also activated microglial cells in the hippocampus, as reflected in bigger soma as well as fewer and shorter branches than normal sleep. Sleep deprivation downregulated phagocytic markers and internalization of postsynaptic protein 95 (PSD95), suggesting impaired synaptic pruning. CX3C motif chemokine receptor 1 (CX3CR1) signaling was detected in in vitro experiments. Sleep deprivation also downregulated CX3CR1. Activation of CX3CR1 signaling increased phagocytosis activity of BV2 microglia in vitro. Sleep deprivation dysregulates microglial CX3CR1 signaling and inhibits synaptic pruning, contributing to associated cognitive deficits. These findings identify CX3CR1-dependent synaptic pruning as a potential therapeutic target in which sleep deprivation causes recognition impairments.

Keywords: CX3CR1; Memory impairments; Microglia; Sleep deprivation; Synaptic pruning.

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Conflict of interest statement

The authors assert that they have no competing financial or personal interests.

Figures

Fig. 1
Fig. 1
Effects of sleep deprivation (SD) on cognitive function in adolescent mice. (a) In the training session, the discrimination index to two similar objects and the training distance were assessed. (b) In the object location test (OLT), the discrimination index of time spent exploring a novel location versus a familiar location and the moving distance was assessed. (c) In the novel object recognition test (NORT), the discrimination index of time spent exploring a novel object versus a familiar object and the moving distance was assessed. (d) In the Y maze, the discrimination index of time spent exploring a novel arm versus a familiar arm and the moving distance were assessed. (e) Body weight was measured daily during the casting session. (f) In the open field test (OFT), the time spent in the center, distance traveled, and time spent moving were assessed. Data are mean ± standard error of the mean (n = 6 animals per condition). *P < 0.05, **P < 0.01, ***P < 0.005.
Fig. 2
Fig. 2
Effects of sleep deprivation (SD) on the phenotype of hippocampal microglia in adolescent mice. (a) Representative morphology of hippocampal microglia in control and sleep-deprived mice. (b) Numbers of Iba1+ cells in the hippocampus. (c) Area of Iba1+ cells in the hippocampus. (d) Numbers of branches on Iba1+ cells in the CA1, CA3, and dentate gyrus (DG) areas of the hippocampus. (e) Lengths of branches on Iba1+ cells in the CA1, CA3, and DG areas. Data are the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005.
Fig. 3
Fig. 3
Effects of sleep deprivation (SD) on microglia-mediated synaptic pruning in adolescent mice. (a) Representative image of microglial phagocytosis of synapses, based on coimmunostaining against Iba1 (red) and PSD95 (green). (b) Comparison of the correlation coefficient of anti-Iba1 and anti-PSD95 immunostaining in the dentate gyrus (DG), CA1, and CA3 areas of the hippocampus. (c) Representative image of microglial lysosomes based on coimmunostaining against Iba1 (red) and CD68 (green). (d) Comparison of the correlation coefficient of Iba1 and CD68 costaining in the dentate gyrus (DG), CA1, and CA3 areas. (e) Levels of PSD95 in the hippocampus. (f) Levels of synapsin in the hippocampus. (g) Numbers of dendritic spines per unit length. Data are the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Effects of sleep deprivation (SD) on CX3CR1 expression in mice and of CX3CR1 exposure on cultures of BV2 cells. (a) Schematic diagram of vitro experiments with BV-2 cells. (b) Alteration in the expression of CX3CR1 when CX3CL1 was administered at different timepoints. (c) Micrograph showing microglia phagocytosing fluorescent microbeads (green) after exposure to CX3CL1. Microglia were immunostained for Iba1 (red). (d) Alterations in the morphology of microglia after exposure to CX3CL1. (e) Alterations in the phagocytic activity of microglia after CX3CL1 treatment at different time points. (f) Hippocampal expression of CX3CR1 at the protein level. (g) Hippocampal expression of TREM2 at the protein level. Data are the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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