Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex

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Abstract

Microglia are the brain’s resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice as compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacologic stimulation of β2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of β2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.

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Fig. 1: Anesthesia enhances microglial surveillance of the parenchyma.
Fig. 2: Microglial surveillance is enhanced by DEX.
Fig. 3: β2-AR signaling reduces microglial dynamics in adolescent mice.
Fig. 4: Inhibition of β2-ARs in awake mice recapitulates the effects of anesthesia by enhancing microglial arborization and surveillance.
Fig. 5: β2-AR activation inhibits microglial process response to focal tissue injury.
Fig. 6: Chronic microglial β2-AR activation impairs adolescent ODP.
Fig. 7: Microglial β2-ARs mediate the effects of clenbuterol on ODP.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

MATLAB code for motility and laser ablation analysis is freely available at https://github.com/majewska-lab. More information can be found in the Life Sciences Reporting Summary.

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Acknowledgements

We thank the University of Rochester Medical Center Flow Core for their expert training and services. We thank C. Lamantia for assistance with animal management; J. Olschowka and K. O’Banion for shared PCR resources; F. Rivera-Escalera for providing training on tissue preparation for microglial FACS; A. Ghosh for the Sholl analysis ImageJ plugin; and J. Cang for sharing MATLAB code for OD analysis. This work was supported by National Institutes of Health grants R01 EY019277 (A.K.M.), R21 NS099973 (A.K.M.), R01 AA027111 (A.K.M.), R01 EY028219 (M.S.), F31 NS105249 (R.D.S.), T32 NS007489 (R.D.S., G.O.S.), F31 NS086241 (G.O.S.) and F32 EY028028 (G.O.S.); National Science Foundation grant NSF 1557971 (A.K.M.); a Schmitt Program on Integrative Brain Research grant (G.O.S. and R.P.D.); the University of Rochester Bilski-Mayer Fellowship (H.N.B.); and the University of Rochester Medical Center Summer Scholars Fellowship (K.A.L.).

Author information

R.D.S., G.O.S. and A.K.M. conceived the project. R.D.S., G.O.S., R.P.D., H.N.B., K.A.L., B.S.W. and M.B.S. carried out experiments and data analysis. R.D.S. carried out iOS imaging experiments and analysis in CX3CR1-CreERT2-AR-flox, CX3CR1-CreERT and C57BL/6J mice, all in vivo two-photon experiments characterizing awake versus anesthetized mice and adrenergic pharmacologic agents, and imaging experiments using CX3CR1-CreERT2-AR-flox/Ai9 and CX3CR1-CreERT/Ai9 mice. R.D.S. also performed all FACS preparation on microglial specimens. G.O.S. carried out iOS imaging experiments in C57BL/6J mice and imaging experiments characterizing stress and circadian rhythms. G.O.S. also performed experiments using terbutaline. G.O.S. performed all resonance imaging and optogenetic experiments. R.P.D. carried out stress experiments. H.N.B. assisted in confirmation of CX3CR1-Cre/β2-AR-flox excision and DSP4 depletion histology experiments. K.A.L. carried out circadian morphology experiments. B.S.W. performed all slice experiments. M.B.S. assisted with DSP4 histology experiments and CX3CR1-Cre/β2-AR-flox expression histology. J.M.B. contributed to the design of experiments with pharmacologic agents. E.B. advised on the design and analysis of stress experiments. M.S. advised on the design and analysis of optogenetic and resonance imaging experiments. R.D.S., G.O.S. and A.K.M. wrote the first draft of the manuscript. All authors contributed to the final version of the manuscript.

Correspondence to Ania K. Majewska.

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Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Figs. 1–15.

Reporting Summary

Supplementary Video 1

Time-lapse movie taken through a chronic cranial window showing the motility of V1 microglia in awake mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 16 mice). Scale bar, 20 μm.

Supplementary Video 2

Time-lapse movie showing the motility of V1 microglia in fentanyl cocktail anesthetized mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time poin,; representative of observations made in n = 16 mice). Scale bar, 20 μm.

Supplementary Video 3

Time-lapse movie taken through a chronic cranial window of V1 microglia in awake mice taken over 1 h at 5-min intervals after focal laser injury (10-μm z stacks were compressed at each time point, representative of observations made in n = 8 mice). Scale bar, 20 μm.

Supplementary Video 4

Time-lapse movie taken through a chronic cranial window of V1 microglia in fentanyl cocktail anesthetized mice taken over 1 h at 5-min intervals after focal laser injury (10-μm z stacks were compressed at each time point, representative of observations made in n = 8 mice). Scale bar, 20 μm.

Supplementary Video 5

Time-lapse movie taken through a chronic cranial window showing the motility of V1 microglia in dexmedetomidine anesthetized mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 7 mice). Scale bar, 20 μm.

Supplementary Video 6

Time-lapse movie taken through a chronic cranial window showing the rapid extension of microglial pseudopodia after dexmedetomidine administration taken with resonance imaging over 45 min at 1.2-s intervals (representative of observations made in n = 7 mice). Scale bar, 10 μm.

Supplementary Video 7

Time-lapse movie taken through an acute craniotomy over V1 showing 30-min baseline microglial motility at 2-min intervals, followed by the retraction of microglial pseudopodia with application of 1-mM terbutaline for 60 min after administration (representative of observations made in n= 4 mice). Scale bar, 20 μm.

Supplementary Video 8

Time-lapse movie taken through a thin-skull preparation showing the motility of V1 microglia in saline dosed mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 16 mice). Scale bar, 20 μm.

Supplementary Video 9

Time-lapse movie taken through a thin-skull preparation showing the motility of V1 microglia in nadolol dosed mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 8 mice). Scale bar, 20 μm.

Supplementary Video 10

Time-lapse movie taken through a thin-skull preparation showing the motility of V1 microglia in nadolol/clenbuterol dosed mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 12 mice). Scale bar, 20 μm.

Supplementary Video 11

Time-lapse movie taken through a thin-skull preparation showing the motility of V1 microglia in DSP4-treated mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 12 mice). Scale bar, 20 μm.

Supplementary Video 12

Time-lapse movie taken through a thin-skull preparation showing the motility of V1 microglia in ICI–118,551-treated mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 11 mice). Scale bar, 20 μm.

Supplementary Video 13

Time-lapse movie taken through a chronic cranial window showing the motility of V1 microglia in awake ICI–118,551-treated mice taken over 1 h at 5-min intervals (30-μm z stacks were compressed in each time point, representative of observations made in n = 7 mice). Scale bar, 20 μm.

Supplementary Video 14

Time-lapse movie taken through a thin-skull preparation of V1 microglia in nadolol-treated mice taken over 1 h at 5 min intervals after focal laser injury (10-μm z stacks were compressed at each time point, representative of observations made in n = 7 mice). Scale bar, 20 μm.

Supplementary Video 15

Time-lapse movie taken through a thin-skull preparation of V1 microglia in nadolol/clenbuterol-treated mice taken over 1 h at 5-min intervals after focal laser injury (10-μm z stacks were compressed at each time point, representative of observations made in n = 5 mice). Scale bar, 20 μm.

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Stowell, R.D., Sipe, G.O., Dawes, R.P. et al. Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex. Nat Neurosci 22, 1782–1792 (2019) doi:10.1038/s41593-019-0514-0

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