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ME Tremblay (2011)16064
J Neurosci, 31
K. Green, Joshua Crapser, Lindsay Hohsfield (2020)To Kill a Microglia: A Case for CSF1R Inhibitors.
Trends in immunology
M. Tremblay, B. Stevens, A. Sierra, H. Wake, A. Bessis, A. Nimmerjahn (2011)The Role of Microglia in the Healthy Brain
The Journal of Neuroscience, 31
Hanxiao Liu, Xinxing Wang, Lu Chen, Liang Chen, S. Tsirka, S. Ge, Q. Xiong (2021)Microglia modulate stable wakefulness via the thalamic reticular nucleus in mice
Nature Communications, 12
H Liu (2021)4646
Nat Commun, 12
Julia Bruttger, K. Karram, Simone Wörtge, Tommy Regen, F. Marini, Nicola Hoppmann, M. Klein, T. Blank, S. Yona, Yochai Wolf, M. Mack, E. Pinteaux, Werner Müller, F. Zipp, H. Binder, T. Bopp, M. Prinz, Steffen Jung, A. Waisman (2015)Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System.
Immunity, 43 1
M. Elmore, A. Najafi, M. Koike, N. Dagher, Elizabeth Spangenberg, Rachel Rice, M. Kitazawa, Bernice Matusow, Hoa Nguyen, B. West, K. Green (2014)Colony-Stimulating Factor 1 Receptor Signaling Is Necessary for Microglia Viability, Unmasking a Microglia Progenitor Cell in the Adult Brain
JP Wisor (2011)261
M. Brancaccio, Mathew Edwards, A. Patton, Nicola Smyllie, J. Chesham, E. Maywood, M. Hastings (2019)Cell-autonomous clock of astrocytes drives circadian behavior in mammals
Giorgio Corsi, K. Picard, M. Castro, S. Garofalo, Federico Tucci, G. Chece, C. Percio, M. Golia, M. Raspa, F. Scavizzi, F. Decoeur, C. Lauro, M. Rigamonti, Fabio Iannello, D. Ragozzino, E. Russo, G. Bernardini, A. Nadjar, M. Tremblay, C. Babiloni, L. Maggi, C. Limatola (2021)Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle
Rocio Barahona, Samuel Morabito, V. Swarup, K. Green (2022)Cortical diurnal rhythms remain intact with microglial depletion
Scientific Reports, 12
L. Fonken, M. Frank, M. Kitt, R. Barrientos, L. Watkins, S. Maier (2015)Microglia inflammatory responses are controlled by an intrinsic circadian clock
Brain, Behavior, and Immunity, 45
Aurélie Brécier, Vina Li, Chloé Smith, Katherine Halievski, N. Ghasemlou (2022)Circadian rhythms and glial cells of the central nervous system
Biological Reviews, 98
Jessica Rosin, D. Kurrasch (2019)Emerging roles for hypothalamic microglia as regulators of physiological homeostasis
Frontiers in Neuroendocrinology, 54
J. Wisor, Michelle Schmidt, William Clegern (2011)Evidence for neuroinflammatory and microglial changes in the cerebral response to sleep loss.
Sleep, 34 3
G Corsi (2022)89
L. Sominsky, Tamara Dangel, S. Malik, S. Luca, N. Singewald, S. Spencer (2020)Microglial ablation in rats disrupts the circadian system
The FASEB Journal, 35
Microglia, as macrophages in the brain, are responsible for immune responses and synaptic remodeling. Although the function of microglia is regulated by circadian rhythms, it is still unclear whether microglia are involved in the generation and light entrainment of circadian rhythms of behavior. Here, we report that microglial depletion does not alter behavioral circadian rhythms. We depleted ~ 95% of microglia in the mouse brain by PLX3397, a CSF1R inhibitor, and analyzed the effect on the spontaneous behaviors of mice. We found that neither the free-running period under constant darkness nor light entrainment under jet-lag circumstances were influenced by the ablation of microglia. Our results demonstrate that the circadian rhythms of locomotor activity, an important output of the circadian clock in the brain, are likely a phenomenon not produced by microglia. Keywords Microglia, Circadian rhythm, Suprachiasmatic nucleus Introduction expressed in roughly 24-hour cycles . Furthermore, Microglia are residential immune cells in the central ner- microglia exhibit circadian rhythm-dependent responses vous system. They are involved in the clearance of apop - to inflammatory stimuli [ 6]. totic cells and synaptic remodeling through phagocytosis u Th s, it is clear that microglia have a close associa - . Recent studies have shown that microglia are closely tion with circadian rhythms as neurons and astrocytes; involved in sleep-wake cycles. For example, depletion however, the evidence supporting the roles of microglia of microglia resulted in the increase of slow-wave sleep on circadian clock system itself, particularly on the loco- (SWS) duration and reduced excitatory neurotransmis- motor activity rhythms, has still been inconclusive [7-9]. sion in the dark period [2, 3]. Another study has reported We re-examined this issue by using the CSF1R inhibi- that microglial depletion suppresses rebound SWS after tor PLX3397 treated mice. Microglia in the adult brain sleep deprivation treatment . Although these studies are fully dependent upon CSF1R signaling for their sur- are partially inconsistent, the results suggest a key role vival. PLX3397 can be administered through food chow for microglia in the sleep/wake homeostasis. with minimal behavioral interference to achieve robust Microglia also have circadian rhythm-related functions. microglial elimination, and so far, no effects on typi - Multiple clock genes and immune activation markers are cal animal behavior and cognitive functions have been reported . Therefore, it is an ideal way to examine the role of microglia in spontaneous animal behavior . *Correspondence: Zhiwen Zhou email@example.com Hiroaki Norimoto firstname.lastname@example.org Graduate School of Medicine, Hokkaido University, Sapporo, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Matsui et al. Molecular Brain (2023) 16:34 Page 2 of 4 Fig. 1 (A) Coronal brain sections from a control mouse (Left) and PLX3397 treated mouse (Right) with Hoechst staining (blue) and microglial marker IBA1 (green) immunolabeling. The dotted line indicates the SCN. Scale bar = 100 μm. (B) PLX3397 treatment induced a 95% microglial depletion in the SCN. N = 7 mice (Control), 8 mice (PLX3397). ***P < 0.001. (C) Mean activity profiles were generated from 7 days in 12 L:12D. N = 12 (Control) and 15 (PLX3397) mice. (D) Analyses of day and night spontaneous locomotor activity counts. (E) Representative double-plotted actograms of control and PLX3397 mice in DD. Shaded gray areas in the actogram represent dark periods. (F) Analyses of spontaneous locomotor activity counts in DD. N = 5 (Control) and 6 (PLX3397) mice. (G) Esti- mated periods (left) and power (right) of circadian rhythms by the Lomb-Scargle periodogram. N = 5 (Control) and 6 (PLX3397) mice. (H) Representative dou- ble-plotted actograms of control and PLX3397 mice subjected to a 13-hour phase advance in LD cycles. (I) Activity onset in the 13-hour phase advance. N = 6 (Control) and 9 (PLX3397) mice. (J) Average interdaily stability from Day 2 to Day 6 (before jet-lag) and from Day 9 to Day 10 (during jet-lag). N = 6 (Control) and 9 (PLX3397) mice. **P < 0.01. (K) Coronal brain section from a control mouse (Left) and a mouse under jet-lag condition (Right) with Hoechst (blue) and microg- lial marker IBA1 (green) immunolabeling. Scale bar = 100 μm. (L) SCN microglial density from the control group and jet-lag group. N = 4 mice for each group. Data are presented as means ± SEM with individual data points plotted. n.s.= non-significant difference. Matsui et al. Molecular Brain (2023) 16:34 Page 3 of 4 Results & discussions the microglia density after experiencing the jet-lag. Mice Mice were randomly assigned to two groups fed with were perfused after three days of LD cycle advancement either a rodent standard chow (control) or chow with a and immunostained for microglia marker IBA1. There CSF1R inhibitor PLX3397, for three weeks. The num - was no difference in microglial density between control ber of microglia in the suprachiasmatic nucleus (SCN), and jet-lag group (Fig. 1K, L, P = 0.73, t = 0.36, Student’s which is well known to generate circadian rhythms, was t-test). significantly reduced in the PLX3397 group (Fig. 1A, B, In the present study, we demonstrated that microg- − 7 P = 2.8 × 10 , t = 9.14, Student’s t-test). Microglia deple- lial depletion does not affect the daily locomotor activ - tion was also observed in other brain regions, consistent ity, free-running rhythms in a DD condition, and the with previous reports (data not shown) . No differ - light entrainment of activity rhythms. These results are ence in locomotor activity was observed between the in a way unexpected because microglia regulate not only control and PLX3397 groups (Fig. 1C, D, P = 0.89, t = higher order brain function in the forebrain but also -0.15 (Light), P = 0.35, t = -0.95 (Dark), Student’s t-test). hypothalamic circuits via the release of inflammatory fac - To examine the effect of microglia depletion on behav - tors and dynamic remodeling of synapses . It should ioral circadian rhythms, we measured animals’ free-run- be noted that these findings are in marked contrast with ning rhythms under constant darkness (DD) condition. previous reports showing that microglial ablation par- The mice were housed in a light controlled home cage tially disrupts the circadian system using Cx3cr1-Dtr with access to food chow and water ad libitum, and their transgenic rats . The contradiction may be explained spontaneous locomotor activity was recorded by accel- by survival rate of microglia, alternative off-target effects erometers. The animals had been receiving the PLX3397 of the ablation method , or distinct functional states treatment for three weeks on day 1 of behavior monitor- of microglia in the different experimental conditions. ing. The mice were housed in a 12-hour light/12-hour Further investigations using functional imaging tech- dark (LD) cycle for one week and then placed in a DD niques and manipulating microglial activity in vivo will condition for two weeks. Both groups exhibited robust help resolve the current controversy regarding the func- rhythms of free-running locomotor activity, and the total tion of microglia on circadian rhythms. locomotor activity did not differ between the control and Abbreviations PLX3397 treated mice during DD condition (Fig. 1E, F, SCN Suprachiasmatic nucleus P = 0.45, t = -0.79, Student’s t-test). The free-running CSF1R Colony stimulating factor 1 receptor SWS Slow wave sleep periods and the power calculated by Lomb-Scargle peri- LD Light/dark odogram also did not differ between the two groups DD Constant darkness (Fig. 1G, P = 0.99, t = -0.02 (periods), P = 0.114, t = 9 9 -0.1139 (power), Student’s t-test). These results imply that microglia do not affect the Supplementary Information The online version contains supplementary material available at https://doi. internal autonomous clock, but it is still possible that org/10.1186/s13041-023-01021-1. microglia function during light entrainment. To test the possibility, we examined the effect of microglia depletion Additional file 1 : Detailed methods. on behavioral rhythms under experimental jet-lag condi- tions. After 1 week of recording behaviors in a normal LD Acknowledgements condition, LD cycles were advanced by 13 h (Fig. 1H). In We thank Dr. Yu Ohmura and Dr. Masaaki Sato for providing the space for experimentation. We thank all members of the Norimoto laboratory for daily both groups, this advance of LD cycles induced a grad- discussions and advice. ual shift of locomotor activity rhythms, which took 5–6 days for the complete re-entrainment to the new LD Author contributions FM, RK, ZZ, and HN designed the study. FM conducted all the surgeries, schedule (Fig. 1H, I). Interdaily stability, a measure of the behavioral experiments, and histological experiments. FM, RK, SY, and ZZ strength of circadian rhythmicity, was reduced during the analyzed the data. RK, SI, and SN helped with data acquisition and analysis. re-entrainment to the new LD cycles (Fig. 1J, Control: FM, ZZ, and HN wrote the manuscript. All authors read and approved the final − 3 manuscript. P = 1.0 × 10 , Q =12.87, Day 2–6 vs. Day 9,10, PLX: 6,6 − 3 P = 1.0 × 10 , Q =19.78, Day 2–6 vs. Day 9,10, Tukey 8,8 Funding test after one-way ANOVA). However, both the onset This work is supported by KAKENHI 22K15369, Sasakawa Scientific Research Grant, Akiyama Research grant, and grant form Hirose Foundation to Z.Z., and timing and the interdaily stability were similar between JST PREST (JPMJPR2048), AMED (22wm0525003s0202), the Murata Science control and microglia depletion groups (Fig. 1I, P = 0.17, Foundation, the Uehara Memorial Foundation, the Mochida Memorial F = 2.08, repeated measure two-way ANOVA; Fig. 1J, 1,1 Foundation for Medical and Pharmaceutical Research, a Grant for Basic Science Research Projects from the Sumitomo Foundation, the Astellas P = 0.59, Q =1.78, Day 9, 10, Control vs. PLX, P = 0.16, 6,8 Foundation for Research on Metabolic Disorders, the Nakajima foundation, the Q =3.08, Day 2–6, Control vs. PLX, Tukey test after 6,8 Naito foundation, and the Toray Science and Technology Grant to H.N. one-way ANOVA). Finally, we examined the changes in Matsui et al. Molecular Brain (2023) 16:34 Page 4 of 4 Data Availability 4. Wisor JP, Schmidt MA, Clegern WC. Evidence for neuroinflammatory The datasets used and/or analyzed for the current study are available from the and microglial changes in the cerebral response to sleep loss. Sleep. corresponding author upon request. 2011;34(3):261–72. 5. Brecier A, Li VW, Smith CS, Halievski K, Ghasemlou N. Circadian rhythms and glial cells of the central nervous system.Biol Rev Camb Philos Soc2022. Declarations 6. Fonken LK, Frank MG, Kitt MM, Barrientos RM, Watkins LR, Maier SF. Microglia inflammatory responses are controlled by an intrinsic circadian clock. Brain Competing interests Behav Immun. 2015;45:171–9. The authors declare no competing interests. 7. Brancaccio M, Edwards MD, Patton AP, Smyllie NJ, Chesham JE, Maywood ES, Hastings MH. Cell-autonomous clock of astrocytes drives circadian behavior Ethics approval and consent to participate in mammals. Science. 2019;363(6423):187–92. All procedures involving the use of animals complied with the guidelines of 8. Sominsky L, Dangel T, Malik S, De Luca SN, Singewald N, Spencer SJ. Microg- the National Institutes of Health and were approved by the Animal Care and lial ablation in rats disrupts the circadian system. FASEB J. 2021;35(2):e21195. Use Committee of the Hokkaido University (Approval numbers:21–0092). 9. Barahona RA, Morabito S, Swarup V, Green KN. Cortical diurnal rhythms remain intact with microglial depletion. Sci Rep. 2022;12(1):114. Consent for publication 10. Elmore MR, Najafi AR, Koike MA, Dagher NN, Spangenberg EE, Rice RA, Not applicable. Kitazawa M, Matusow B, Nguyen H, West BL, et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia Received: 14 February 2023 / Accepted: 19 March 2023 progenitor cell in the adult brain. Neuron. 2014;82(2):380–97. 11. Green KN, Crapser JD, Hohsfield LA. To kill a Microglia: a case for CSF1R inhibi - tors. Trends Immunol. 2020;41(9):771–84. 12. Rosin JM, Kurrasch DM. Emerging roles for hypothalamic microglia as regula- tors of physiological homeostasis. Front Neuroendocrinol. 2019;54:100748. 13. Bruttger J, Karram K, Wortge S, Regen T, Marini F, Hoppmann N, Klein M, References Blank T, Yona S, Wolf Y, et al. Genetic cell ablation reveals clusters of local self- 1. Tremblay ME, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A. The role of renewing Microglia in the mammalian Central Nervous System. Immunity. microglia in the healthy brain. J Neurosci. 2011;31(45):16064–9. 2015;43(1):92–106. 2. Corsi G, Picard K, di Castro MA, Garofalo S, Tucci F, Chece G, Del Percio C, Golia MT, Raspa M, Scavizzi F, et al. Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle. Glia. Publisher’s Note 2022;70(1):89–105. Springer Nature remains neutral with regard to jurisdictional claims in 3. Liu H, Wang X, Chen L, Chen L, Tsirka SE, Ge S, Xiong Q. Microglia modulate published maps and institutional affiliations. stable wakefulness via the thalamic reticular nucleus in mice. Nat Commun. 2021;12(1):4646.
Molecular Brain – Springer Journals
Published: Apr 7, 2023
Keywords: Microglia; Circadian rhythm; Suprachiasmatic nucleus
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