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We previously found that Mertk and its ligand Gas6, astrocytic genes involved in phagocytosis, are upregulated after acute sleep deprivation. These results suggested that astrocytes may engage in phagocytic activity during extended wake, but direct evidence was lacking. Studies in humans and rodents also found that sleep loss increases peripheral markers of inflammation, but whether these changes are associated with neuroinflammation and/or activation of microglia, the brain's resident innate immune cells, was unknown. Here we used serial block-face scanning electron microscopy to obtain 3D volume measurements of synapses and surrounding astrocytic processes in mouse frontal cortex after 6–8 h of sleep, spontaneous wake, or sleep deprivation (SD) and after chronic (∼5 d) sleep restriction (CSR). Astrocytic phagocytosis, mainly of presynaptic components of large synapses, increased after both acute and chronic sleep loss relative to sleep and wake. MERTK expression and lipid peroxidation in synaptoneurosomes also increased to a similar extent after short and long sleep loss, suggesting that astrocytic phagocytosis may represent the brain's response to the increase in synaptic activity associated with prolonged wake, clearing worn components of heavily used synapses. Using confocal microscopy, we then found that CSR but not SD mice show morphological signs of microglial activation and enhanced microglial phagocytosis of synaptic elements, without obvious signs of neuroinflammation in the CSF. Because low-level sustained microglia activation can lead to abnormal responses to a secondary insult, these results suggest that chronic sleep loss, through microglia priming, may predispose the brain to further damage.
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Sleep Loss Promotes Astrocytic Phagocytosis and Microglial
Activation in Mouse Cerebral Cortex
XMichele Bellesi,
Luisa de Vivo,
XMattia Chini,
Francesca Gilli,
Giulio Tononi,
and XChiara Cirelli
Department of Psychiatry, University of Wisconsin–Madison, Madison, Wisconsin 53719,
Department of Experimental and Clinical Medicine, Section of
Neuroscience and Cell Biology, Universita` Politecnica delle Marche, Ancona, 60026, Italy, and
Department of Neurology, Geisel School of Medicine at
Dartmouth, Lebanon, New Hampshire 03756
We previously found that Mertk and its ligand Gas6, astrocytic genes involved in phagocytosis, are upregulated after acute sleep depri-
vation. These results suggested that astrocytes may engage in phagocytic activity during extended wake, but direct evidence was lacking.
Studies in humans and rodents also found that sleep loss increases peripheral markers of inflammation, but whether these changes are
associated with neuroinflammation and/or activation of microglia, the brain’s resident innate immune cells, was unknown. Here we used
serial block-face scanning electron microscopy to obtain 3D volume measurements of synapses and surrounding astrocytic processes in
mouse frontal cortex after 6 –8 h of sleep, spontaneous wake, or sleep deprivation (SD) and after chronic (5 d) sleep restriction (CSR).
Astrocytic phagocytosis, mainly of presynaptic components of large synapses, increased after both acute and chronic sleep loss relative
to sleep and wake. MERTK expression and lipid peroxidation in synaptoneurosomes also increased to a similar extent after short and long
sleep loss, suggesting that astrocytic phagocytosis may represent the brain’s response to the increase in synaptic activity associated with
prolonged wake, clearing worn components of heavily used synapses. Using confocal microscopy, we then found that CSR but not SD mice
show morphological signs of microglial activation and enhanced microglial phagocytosis of synaptic elements, without obvious signs of
neuroinflammation in the CSF. Because low-level sustained microglia activation can lead to abnormal responses to a secondary insult,
these results suggest that chronic sleep loss, through microglia priming, may predispose the brain to further damage.
Key words: astrocyte; cortex; microglia; mouse; sleep; sleep deprivation
Astrocytes are influenced by changes in behavioral state. Using
serial block-face scanning electron microscopy (SBEM), we re-
cently found that most excitatory synapses in mouse frontal cor-
tex are contacted by peripheral astrocytic processes (PAPs). PAPs
move closer to the synaptic cleft and expand after extended wake,
presumably because the need to clear glutamate and potassium
ions increases. Transcriptomic profiling also showed that the ex-
pression of 1.4% of astrocytic genes is state dependent and
mostly upregulated in wake relative to sleep (Bellesi et al., 2015).
Astrocytic “wake” genes in mice included Mertk (Bellesi et al.,
2015), and previous experiments in rats found that Gas6 was
upregulated in cortex after chronic sleep deprivation (Cirelli et
Received Dec. 31, 2016; revised March 23, 2017; accepted April 13, 2017.
Author contributions: M.B., G.T., and C.C. designed research; M.B., L.d.V., and M.C. performed research; M.B.,
L.d.V., M.C., and F.G. analyzed data; M.B., G.T., and C.C. wrote the paper.
This work was supported by NIH Grants DP 1OD579 (G.T.), 1R01MH091326 (G.T.), 1R01MH099231 (G.T., C.C.),
and 1P01NS083514 (G.T., C.C.). We thank Benjamin Jones, Hirotaka Nagai, Midori Nagai, Sakiko Honjoh, Alex
Rodriguez, Kayla Peelman, Douglas Haswell, and Giovanna Spano for helping with the chronic sleep restriction
experiments and Sophia Loschky, Andrea Schroeder, and Samuel Koebe for contributions to EM image analysis.
The authors declare no competing financial interests.
Correspondence should be addressed to Chiara Cirelli, Department of Psychiatry, University of Wisconsin–
Madison, 6001 Research Park Boulevard, Madison, WI 53719. E-mail:
Copyright © 2017 the authors 0270-6474/17/375263-11$15.00/0
Significance Statement
We find that astrocytic phagocytosis of synaptic elements, mostly of presynaptic origin and in large synapses, is upregulated
already after a few hours of sleep deprivation and shows a further significant increase after prolonged and severe sleep loss,
suggesting that it may promote the housekeeping of heavily used and strong synapses in response to the increased neuronal
activity of extended wake. By contrast, chronic sleep restriction but not acute sleep loss activates microglia, promotes their
phagocytic activity, and does so in the absence of overt signs of neuroinflammation, suggesting that like many other stressors,
extended sleep disruption may lead to a state of sustained microglia activation, perhaps increasing the brain’s susceptibility to
other forms of damage.
The Journal of Neuroscience, May 24, 2017 37(21):5263–5273 • 5263
al., 2006). The receptor MERTK belongs to one of the two path-
ways that mediate astrocytic phagocytosis (AP; Chung et al.,
2015) and through the action of GAS6 (growth arrest-specific
protein 6) binds exposed phosphatidylserine in target debris
(Grommes et al., 2008). AP participates in developmental synap-
tic pruning (Berbel and Innocenti, 1988;Chung et al., 2013) and
adult astrocytes engulf axonal organelles and synaptic elements
even in healthy mice, suggesting that their constitutive phagocytic
activity contributes to the clearing of damaged cellular components
(Nguyen et al., 2011;Chung et al., 2013;Davis et al., 2014), likely in
response to wake-related neuronal activity (Chung et al., 2015).
Microglia are the resident phagocytes of the CNS. They con-
stantly monitor the surrounding microenvironment via their
processes, sense neuronal activity, clear neuronal debris after in-
jury and cell death (Wake et al., 2009;Tremblay et al., 2010;Tay et
al., 2017), and contribute to developmental synaptic pruning in
the healthy brain (Paolicelli et al., 2011;Schafer et al., 2012;Bialas
and Stevens, 2013;Sipe et al., 2016). Microglial phagocytosis is
mediated by C1q and C3, components of the complement cas-
cade that tag unwanted synapses; by the phagocytic complement
receptor expressed by microglia (Stevens et al., 2007); and by
MERTK, which is also expressed in microglia (Chung et al., 2013).
Any disturbance of brain homeostasis, including inflammation,
activates microglia. Acute and chronic sleep deprivation can lead
to a pro-inflammatory state in the absence of overt infection or
injury (Mullington et al., 2010;Hurtado-Alvarado et al., 2013).
Specifically, in humans and rodents, sleep loss can lead to ele-
vated white blood cell counts; increased circulating levels of
C-reactive protein, IL1
, IL6, and TNF
(Everson, 2005;Mull-
ington et al., 2010;Hurtado-Alvarado et al., 2013;He et al., 2014);
and enhanced permeability of the blood– brain barrier (Hurtado-
Alvarado et al., 2013;He et al., 2014). The source of the increase in
peripheral cytokines remains unclear but has been linked to the
increase in catecholamine levels associated with prolonged wake
(Mullington et al., 2010). Equally unclear is whether these pe-
ripheral changes are associated with signs of neuroinflammation
and/or with microglial activation.
Together, these findings suggest that sleep loss can trigger AP
and lead to microglia activation. Here we tested this hypothesis
using SBEM to study PAPs surrounding cortical mouse synapses
and measured AP occurrence after sleep, spontaneous wake, and
sleep loss. In cortical synaptoneurosomes, we also assessed changes
in MERTK protein levels and the extent of lipid peroxidation,
which can result from high oxidative stress and in turn can trig-
ger phagocytosis. In addition, we measured microglia state of
activation and phagocytic activity, as well as levels of inflam-
matory markers in the CSF of mice after sleep and sleep loss. We
find that AP, mainly of presynaptic elements in large synapses,
occurs after both acute and chronic sleep loss but not after spon-
taneous wake, suggesting that it may promote the housekeeping
and recycling of worn components of heavily used, strong syn-
apses. By contrast, only chronic sleep loss activates microglia cells
and promotes their phagocytic activity, apparently without overt
signs of neuroinflammation, suggesting that extended sleep dis-
ruption may prime microglia and perhaps predispose the brain to
other forms of insult.
Materials and Methods
Four-week-old homozygous B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic
mice of either sex were used in this study with the exception of microglia
experiments, in which 4-week-old male C57BL/6J mice were used. Mice
were housed in recording boxes for the duration of the experiment (12 h
light/dark cycle, light on at 8:00 A.M., 23 1°C; food and water available
ad libitum and replaced daily at 8:00 A.M.). All animal procedures fol-
lowed the National Institutes of Health Guide for the Care and Use of
Laboratory Animals, and facilities were reviewed and approved by the
Institutional Animal Care and Use Committee of the University of
Wisconsin–Madison and were inspected and accredited by the Associa-
tion for Assessment and Accreditation of Laboratory Animal Care.
Experimental conditions and protocols for sleep, spontaneous
wake, and acute and chronic sleep loss
Four experimental conditions were used (Fig. 1A): (1) sleeping (S) mice
were killed during the light phase, at the end of a long period of sleep
(45 min, interrupted by periods of wake of 4 min), and after spending
at least 75% of the previous 6 –7 h asleep; (2) spontaneously awake (W)
mice were killed during the dark phase, at the end of a long period of wake
(1 h, interrupted by periods of sleep of 5 min), and after spending at
least 70% of the previous 6 –7 h awake; (3) acutely sleep-deprived (SD)
mice were killed during the light phase after8hofsleep deprivation
enforced by introducing novel objects and by tapping on the cage when-
ever the animals appeared drowsy. As demonstrated in previous studies
with EEG recordings, this method can prevent sleep almost completely
for several hours [95% total time spent awake (Cirelli et al., 2004;
Bellesi et al., 2013,2015)]; and (4) chronically sleep-restricted (CSR)
mice were subjected in groups (six to eight mice per group) to 4.5 days of
chronic sleep restriction using a protocol optimized in our laboratory. A
previous validation study that used mice implanted with EEG electrodes
found that this CSR protocol results in a reduction of overall sleep dura-
tion by 70% (de Vivo et al., 2016). During chronic sleep restriction,
mice were housed in large cages where, during the day, sleep restriction
was enforced using ecologically relevant stimuli that included continuous
exposure to novel objects, changes of cage and bedding, social interaction,
and free access to multiple running wheels. Mild forced locomotion on a
slowly rotating platform was used to restrict sleep at night. The platform
was located above a tray filled with 2–3 cm of water, and the rotation
speed was low enough that mice (still in groups) could easily avoid falling
into the water if they moved continuously. Heat lamps were placed 2m
above the platform to keep mice at the proper temperature. Video cam-
eras and/or direct visual observation were used to continuously monitor
the mice. If a mouse fell in the water often enough such that it did not
have a chance to dry, it was removed to a cage filled with novel objects and
allowed to dry before being placed back onto the rotating platform. Be-
cause CSR mice were in groups, sleep and wake could not be quantified
with video recording as in S and W mice (see below).
Experimental procedure
S, W, and SD mice were individually housed starting at postnatal day 27
(P27) and killed at P30, and all of them were exposed to a few novel
objects and had access to running wheels during the dark phase. CSR
mice were subjected to sleep restriction in groups (six to eight mice per
group) from P25 to P30. S, SD, and CSR mice were killed at the same time
of day (4:00 P.M.), whereas W mice were killed at 4:00 A.M. (Fig.
1A). Independent groups of S, SD, and CSR mice were used for ultra-
structural, molecular, and histological studies (W mice were used only
for the ultrastructural studies).
Video recordings of behavioral states. To avoid possible tissue damage
and inflammation resulting from the implant of EEG electrodes, behav-
ioral states in S and W mice were determined by continuous video mon-
itoring with infrared cameras. As described previously (Maret et al., 2011;
Bellesi et al., 2015), this method consistently estimates total sleep time
with 90% accuracy even if it cannot distinguish nonrapid eye move-
ment sleep from rapid eye movement sleep. Motor activity was then
quantified by custom-made video-based motion detection algorithms
(Bellesi et al., 2012).
Ultrastructural studies. The image dataset used for the ultrastructural
analysis of astrocytic endocytosis is the same used in a previous study that
characterized the dynamics of peripheral astrocytic processes (PAPs) in
S, W, SD, and CSR mice (Bellesi et al., 2015), and a detailed description of
the methods (perfusion, staining, acquisition, and profiles segmenta-
tion) is reported there. Briefly, mice (three animals per group) were perfused
5264 J. Neurosci., May 24, 2017 37(21):5263–5273 Bellesi et al. Glial Phagocytosis in Sleep and Wake
under deep anesthesia (3% isoflurane). Tissue was stained with a solution of
1.5% potassium ferrocyanide/2% osmium tetroxide, followed by 1% thio-
carbonhydrazide, 2% osmium tetroxide, and 1% uranyl acetate at 4°C. The
following day, the tissue was stained with a solution of lead aspartate, dehy-
drated, and embedded with Durcupan resin and ACLAR film. Small squares
of tissue (1 mm
) from frontal cortex (anteroposterior, 1.85 mm; mediolat-
eral, 1.5 mm) were glued on the tip of a metal pin and coated with silver paint
to minimize specimen charging during imaging.
Image acquisition. Images were obtained using a IGMA VP field emis-
sion scanning electron microscope (Carl Zeiss) equipped with 3View tech-
nology (Gatan) and a backscattered electron detector (for SBEM). The
series of images were processed and analyzed using TrakEM2, a FIJI
plug-in (Schindelin et al., 2012). Segmentation of astrocytic profiles was
performed manually by two operators blind to the experimental condi-
tion. Small cuboid regions of interest (ROIs; 5– 6
m per side) of neuro-
pil (layers II–III, frontal cortex) were selected. PAPs were recognized
based on their distinctive shapes, interdigitating among neuronal profiles
and often contacting parts of the synapse, and on the presence of glyco-
gen granules. ROIs did not include large dendrites or somata of neurons,
glia, or endothelial cells. For each ROI, astrocytic volume and ROI vol-
ume were estimated. The occurrence of AP was established by the pres-
ence of a portion of axon, spine head, or dendrite being invaginated by
Figure 1. Sleep loss promotes AP. A, Experimental design. B, Volume of all ROIs analyzed in S (n295), W (n266), SD (n355), and CSR (n280) mice. Black bars depict mean and SD.
C, Example of AP as visualized in two-dimensional SBEM images (left) and its 3D reconstruction (right). Scale bar: 200 nm. D, Left, Number of synaptic elements phagocyted by astrocytes in S, W, SD,
andCSR mice. Values (meanSEM) areexpressed percubed millimeter ofastrocytic volume.*p0.05; **p0.01;***p0.001. Right,Breakdown frequencyanalysis of theneuropil structures
involved in AP for S, W, SD, and CSR. E, ASI size of all S, W, SD, and CSR APsynapses relative to mean ASI size (dashed line) in a random sample of synapses (S, n302; W, n256; SD, n345;
CSR, n296). F, Example of a presynaptic bouton (yellow) containing a mitochondrion (asterisk) and being phagocyted by a PAP (blue). Scale bar, 400 nm. G, Percentage of presynaptic boutons
containing a mitochondrion that are (blue bars) or are not (green bars) involved in AP in S, W, SD, and CSR mice. H, Examples of FE (asterisk, left) and EE (asterisk, right). Scale bar, 130 nm. I,3D
reconstruction of one EE (red). Note its tubular structure within the PAP (light blue). J, Number of EE and FE (mean SEM) per cubed millimeter of astrocytic volume in S, W, SD, and CSR mice.
Bellesi et al. Glial Phagocytosis in Sleep and Wake J. Neurosci., May 24, 2017 37(21):5263–5273 • 5265
the surrounding PAP, with a clear continuity between the part being
enclosed by the PAP (phagosome) and the neuronal structure. AP was
quantified using the following score: 1, phagocytosis of the spine head; 2,
phagocytosis of the presynaptic bouton; 3, phagocytosis of the axon (out-
side the axonal bouton); 4, phagocytosis of the dendritic shaft; 5, phago-
cytosis of an unknown structure. For those synapses whose axonal
bouton or spine head were involved in AP, the axon–spine interface
(ASI) was manually segmented and measured [as in the study by Bellesi et
al. (2015)]. Inside the PAPs, endosomes showing undigested, partially, or
fully digested material were scored as full endosomes (FE), whether or
not they were fused with lysosomes, whereas the endosomes with no
vesicles were scored as empty endosomes (EE).
Microarray: data analysis
We used the microarray data available at the NCBI Gene Expression
Omnibus (GEO) database (GSE60079) to perform gene expression anal-
ysis of cerebral cortex samples collected from sleeping (6 –7 h of sleep
during the light phase), awake (6 –7 h of spontaneous wake at night), and
forced enriched wake (4 h of sleep deprivation through exposure to novel
objects during the light phase) mice. Detailed methods were described by
Bellesi et al. (2015). Briefly, samples (six for each behavioral state) were
collected using the genetically targeted translating ribosome affinity pu-
rification methodology from bacterial artificial chromosome transgenic
mice expressing EGFP-tagged ribosomal protein L10a in astrocytes.
Samples were immunoprecipitated to isolate astrocytes. The precipitated
portion formed the bound (IP) sample containing astrocytes, and the
remaining part formed the unbound (UB) sample containing all the
remaining cell types (neurons and other glia cells). Then, both IP and UB
samples were processed, and RNA was extracted and run on Affymetrix
GeneChip Mouse Genome 430 2.0 arrays. In the present study, we used
array data obtained from the IP samples, and we compared S versus W
and S versus SD mice. Data were normalized within each behavioral state
group using Robust Multiarray Average. Comparisons were performed
using Welch’s ttest with Benjamini and Hochberg FDR multiple-test
correction. All probe sets with fold change 30% and p0.01 were
considered as differentially expressed.
Synaptoneurosome preparation and Western blotting. Under anesthesia,
mice (four S, four SD, four CSR) were decapitated, and the cerebral
cortex (including the striatum) was quickly collected. Samples were ho-
mogenized in ice-cold homogenization buffer [10 mMHEPES (Sigma),
1.0 mMEDTA (Promega), 2.0 mMEGTA (Thermo Fisher Scientific),
0.5 mMDTT (Invitrogen), 0.1 mMPMSF (Fluka), 10 mg/L leupeptin
(Sigma), 50 mg/L soybean trypsin inhibitor (Roche), and 100 nMmicro-
cystin (Alexis)] using a glass/glass tissue homogenizer (Kontes). A frac-
tion (10%) of the homogenate from each sample was boiled in 10%
SDS for 10 min and stored unprocessed at 80°C. The remaining frac-
tion of the homogenate was passed through two 105
m pore nylon mesh
filters (Small Parts), then through a 5
m pore filter (Millipore), and
centrifuged at 1000 gfor 10 min at 4°C. Pellets were resuspended in 1%
SDS, boiled for 10 min, and stored at 80°C. Protein concentration was
determined by the bicinchonic acid assay (Pierce). Since housekeeping
proteins (e.g.,
-actin and
-tubulin) can be affected by sleep and wake,
they were not used as an internal standard. Instead, for both homoge-
nates and synaptoneurosomes, equal amounts of protein were pooled
from each individual animal within each group. S, SD, and CSR pools
(four mice per group) were loaded onto the same gels in three to six
replicates (sample loading was randomized). The entire procedure, from
pool preparation to sample loading, was repeated four times. In each
experiment, equal amounts (5
g for GFAP, 10
g for MERTK, 20
g for
C3) of homogenate/synaptoneurosome from S, SD, and CSR pools were
separated by Tris-HCl gel electrophoresis (Bio-Rad). Nitrocellulose
membranes were probed with anti-GFAP (1:500, Sigma), anti-MERTK
(1:500, R&D Systems; AF591), or anti-C3 (1:500, Cappel Laboratories)
antibodies. After exposure to secondary antibodies, bands were visual-
ized using enhanced chemiluminescence (ECL-Prime, GE Healthcare)
and captured by the Typhoon 9410 Variable Mode Imager (GE Health-
care). Optical densities were calculated for each band of interest after
performing background correction (by subtracting the value of a band
immediately above the band of interest in the same lane) and normalized
within each experiment to the average density of S samples.
Lipid peroxidation. Lipid peroxidation was evaluated in cortical synap-
toneurosomes of S (n7), SD (n8), and CSR (n7) mice using the
Lipid Peroxidation (MDA) Assay kit (ab118970, Abcam). This assay pro-
vides an estimation of the end product [malondialdehyde (MDA)] of
lipid peroxidation. Aliquots of synaptoneurosomes (200
l) were incu-
bated with thiobarbituric acid (TBA) at 95°C for 60 min to generate a
MDA–TBA adduct, which was quantified colorimetrically (OD, 532 nm)
using a microplate reader.
CSF extraction and Luminex multiplex immune assay. Under anesthe-
sia, S (n11), SD (n10), and CSR (n8) mice were placed on a
stereotaxic apparatus, meninges overlying the cisterna magna were ex-
posed, and the surrounding area was gently washed to prevent blood
contamination. A small glass capillary tube was used to puncture the
arachnoid membrane covering the cisterna magna and collect CSF by
capillary action. Approximately 10
l of CSF were obtained from each
mouse and immediately stored at 80°C. Cytokine and chemokine con-
centrations were measured in a multiplex Luminex assay, i.e., the Bio-
Plex Pro Mouse Cytokine 23-plex Assay (Bio-Rad). Individual CSF
samples were diluted to 50
l of volume and incubated with a suspension
of analyte capture antibody-conjugated microspheres, per the manufac-
turer’s instructions. After further incubation with biotinylated detection
antibodies and phycoerythrin (PE)-conjugated streptavidin, fluorescent
signal was read on a Luminex MAGPIX Multiplex Reader (Bio-Rad). A
five-parameter logistic curve generated from standards of known con-
centration was used to convert fluorescent intensity to concentration
values, which were then adjusted for sample dilution. The analyzed mol-
ecules were IL1a, IL1b, IL2, IL3, IL4, IL5, IL6, IL9, IL10, IL12(p40),
IL12(p70), IL13, IL17a, Eotaxin, G-CSF, GM-CSF, IFN
, KC, MCP1,
Immunocytochemistry. S(n6), SD (n5), and CSR (n6) mice
were deeply anesthetized with isoflurane (1–1.5% volume) and perfused
transcardially with a flush (30 s) of saline, followed by 4% paraformal-
dehyde in phosphate buffer. Brains were removed, postfixed in the same
fixative overnight, and cut on a vibratome in 50
m coronal sections.
Sections were rinsed in a blocking solution [3% bovine serum albumin
(BSA) and 0.3% Triton X-100 for IBA-1 and V-GLUT1, 2% BSA and
0.2% Triton X-100 for MERTK] for 1 h and incubated overnight (4°C) in
the same blocking solution containing anti-IBA-1 (1:500, catalog #019-
19741, Wako), anti-VGLUT-1 (1:1000, ab5905, Millipore), or anti-
MERTK (1:100, AF591, R&D Systems). Sections were then probed with
secondary antibodies: Alexa Fluor 568 (1:500, Invitrogen)- and/or Alexa
Fluor 488 (1:500, Invitrogen)-conjugated secondary antibodies. For
MERTK staining, signal was amplified using anti-goat biotinylated
antibodies (1:100, Vector Laboratories) and the TSA kit #22, with HRP–
streptavidin and Alexa Fluor 488 Tyramide (T-20932); after the amplifi-
cation, sections were incubated with anti-GFAP antibodies (1:100,
Sigma; overnight at 4°C) and probed with Alexa Fluor 568 (1:500, Invit-
rogen). Sections were examined with a confocal microscope (Prairie
Technologies). For IBA-1, microscopic fields (n5 per section, 3 sec-
tions per mouse) were randomly acquired as 512 512 pixel images
(pixel size, 581 nm; Z-step, 750 nm) in mouse frontal cortex using a
UPlan FL N 40objective (numerical aperture, 1.3). To improve the
signal/noise ratio, two frames of each image were averaged. For IBA-1/
VGLUT-1, microscopic fields (n5 per section, 3 sections per mouse)
were randomly acquired as 1024 1024 pixel images (pixel size, 65 nm;
digital zoom, 3) in mouse frontal cortex using a UPlan FL N 60
objective (numerical aperture, 1.3).
Image analysis. For IBA-1 staining, all analyses were performed on
maximum-intensity projections (Z-project, Maximum Intensity func-
tion in ImageJ) of the 28 images constituting the Z-stack. Cell counting
was performed manually by two operators blind to the experimental
conditions using the cell-counting plugin of FIJI. Two methods were
implemented for the morphological analysis of microglia.
Method 1 was adapted from Morrison and Filosa (2013) and consisted
on the skeleton analysis of microglial processes. Briefly, background noise of
Z-projected images was diminished using the function “Despeckle” in FIJI.
5266 J. Neurosci., May 24, 2017 37(21):5263–5273 Bellesi et al. Glial Phagocytosis in Sleep and Wake
Then, images were binarized, skeletonized, and analyzed using the FIJI
plugin “Analyze Skeleton.” Branch length and the number of end points
(extremities) were then divided by the number of cell somas per frame to
obtain normalized values. Method 2 was adapted from Kozlowski and
Weimer (2012): images were first thresholded using the “Graythresh”
function within MATLAB, and objects with a size comprised between
200 and 1500 pixels, corresponding to putative microglial cells, were
identified. To analyze individual cells, the centroid (center of mass) for
each of these objects was computed and used to crop an ROI of 110 110
pixels around each cell. Each cell mask was visually examined to confirm
that a single microglial cell was accurately represented in the mask. Im-
ages in which the cell touched the boundary of the image, or images that
did not contain a single cell soma, were not considered for further
analysis. Overall, 5129 of 8653 microglial cells met the inclusion cri-
teria and were subsequently analyzed using the function “Regionprops”
in MATLAB to obtain an estimation of cell perimeter and area. Microglia
phagocytosis was quantified in the double-stained IBA-1/VGLUT-1 im-
ages. To optimize the detection of the VGLUT-1-positive puncta en-
gulfed within the microglia, green (IBA-1) and red (VGLUT-1) channels
were processed separately. The background noise of the green channel
was reduced by using the function Subtract Background (rolling ball
radius, 50 pixels) in FIJI. The image was subsequently filtered using a 3D
hysteresis filter1, followed by a 3D median filter2 in Matlab. The back-
ground noise of the red channel was diminished using the function Sub-
tract Background (rolling ball radius, 2 pixels) and Despeckle in FIJI. The
image was subsequently filtered through a 3D Maximum Filter (radius, 3
pixels in every dimension), automatically thresholded (“Auto Threshold”,
“Default” method), and segmented using the “Watershed” function. Green
and red channels were then remerged. Only VGLUT-1-positive puncta big-
ger than 100 pixels (0.03
)inxyz, and showing 100% overlap with the
processed IBA-1 signal, were quantified.
Sleep loss enhances astrocyte phagocytosis
To study the occurrence of astrocytic phagocytosis in the cortical
neuropil, tridimensional ROIs were manually segmented and an-
alyzed in layers II/III of the mouse frontal cortex, in mice that
slept, were spontaneously awake, or were acutely or chronically
deprived of sleep (Fig. 1A; three mice per group; number of ROIs:
S, 295; W, 266; SD, 355; CSR, 280). The amount of analyzed
neuropil was similar across conditions [1mm
; Kruskal–Wallis
(KW) test, p0.45; Fig. 1B]. The four groups also did not differ
in analyzed astrocytic volume (KW test, p0.11, data not
shown), nor in mean synaptic density per ROI (number of syn-
apses/ROIs: S, 2.81; W, 3.03; SD, 2.93; CSR, 2.74). PAPs were
easily recognized because of their morphological features (see
Materials and Methods), and inside PAPs, AP was identified
structurally by the presence of a portion of spine head, axon, or
dendrite surrounded by the PAP, with a clear continuity between
the part being enclosed by the PAP (phagosome) and the neuro-
nal structure (see Fig. 1Cfor an example). Cumulative distribu-
tion analysis of all listed ROIs (S: n289; W: n266; SD: n
355; CSR: n280) showed that in all mice AP affected only a
small minority of synapses, but it changed across the experimen-
tal conditions (KW test, p0.0001). Specifically, AP occurred
more frequently in CSR and SD mice than in S mice (percentage
of all synapses within the ROI: CSR, 13.5%; W, 7.3%; SD, 8.4%; S,
5.7%; Dunn’s multiple comparison test, CSR vs S, p0.0001; SD
vs S, p0.0076; CSR vs SD, p0.026; Fig. 1D). In addition, we
found that AP occurrence in W mice was comparable to S mice
(Dunn’s multiple comparison test, p0.12) and significantly
different from CSR mice (Dunn’s multiple comparison test, p
0.005) but not from SD mice (Dunn’s multiple comparison test,
p0.9; Fig. 1D), suggesting that the increase in AP was related to
sleep loss and not just to the wake state. Note that the increase in
AP after sleep loss is unlikely to be explained by the exposure to
novel objects and running wheels during acute and chronic sleep
deprivation, because S and W mice were also exposed to the same
stimuli during the dark phase. Further analysis of the specific
structures of neuropil involved in AP revealed that axons and
axonal boutons accounted for 75% of all phagocyted elements,
and spine heads for 18 –20% of all phagocyted elements (Fig.
1D). Components that could not be identified were rare, and
parts of the dendritic shafts were almost never seen (Fig. 1D).
Despite the change in absolute number of synapses involved in
AP (APsynapses), the proportion of axons plus boutons rela-
tive to spine heads was primarily maintained across the four con-
ditions (S, W, SD, CSR), suggesting that sleep loss promotes AP as
a whole, with no specific effects on select components of the
During early development, AP mediates the elimination of
weak synapses in the lateral geniculate nucleus (Chung et al.,
2013). Given the correlation between synaptic strength and size
(Holtmaat and Svoboda, 2009), we measured the size of AP
synapses to test whether small synapses were more frequently
phagocyted by PAPs. We considered all synapses whose axon
bouton or spine head was being phagocyted and measured their
ASI, a reliable measure of synaptic strength that is also highly
correlated with spine head volume (Desmond and Levy, 1988).
AP involving other components outside the synapse (axons, den-
drites, and unknown) was not considered in this analysis. We
found that in all groups, the ASI of APsynapses was larger than
the average ASI size [Mann–Whitney U(MW) test, p0.01]
calculated from a pool of synapses randomly chosen in the S (n
302 synapses), W (n256), SD (n345), and CSR (n296)
datasets. Thus, independent of behavioral state, large synapses
were more likely to show AP than synapses of medium or small
size. We also quantified the prevalence of axonal boutons con-
taining one or more mitochondria in APand APsynapses of
comparable size (mitochondria are rare in spine heads; Sorra and
Harris, 2000). As before, APsynapses were selected from a pool
of synapses randomly chosen from the S (n216 synapses), W
(n206), SD (n301), and CSR (n203) datasets. In S and SD
mice, a strong trend toward an increase in the number of axonal
boutons with mitochondria was seen in APsynapses relative to
APsynapses (APvs AP: S, 57.2% vs 36.6%; SD, 50% vs
39.2%). However, Fisher’s exact test did not reach significance (S,
p0.098; SD, p0.22), and no changes were observed in the W
(APvs AP: W, 45.7% vs 43.7%; p0.86) and CSR (APvs
AP: 38.3% vs 40.9%; p0.87; Fig. 1G) groups.
In 30% of ROIs, PAPs contained endosomes, defined as
cytoplasmic membranous organelles of various size. To verify
whether their number was affected by experimental condition,
we annotated their presence while assessing AP. Since it was very
difficult to visually distinguish endosomes based on the different
types of inclusions, such as undigested engulfed synaptic material
or partially digested material, we considered all endosomes con-
taining some material as FE (Fig. 1H, left), whereas endosomes
with no material were scored as EE (Fig. 1H, right). Notably,
often EE appeared to form a complex tubular structure within
the PAP in the 3D reconstruction (Fig. 1I). Although the num-
ber of FE did not change significantly across experimental
conditions (KW test, p0.13), the number of EE showed a
large increase in S mice relative to SD (MW test, p0.0001)
and CSR (MW test, p0.0001) mice. The density of EE in W
mice was different from S mice (MW test, p0.045) but also
from SD (MW test, p0.006) and CSR (MW test, p0.0002)
mice (Fig. 1J).
Bellesi et al. Glial Phagocytosis in Sleep and Wake J. Neurosci., May 24, 2017 37(21):5263–5273 • 5267
Sleep loss increases the expression of MERTK
In a recent study in Aldh1L1-eGFP-L10a mice, we used translat-
ing ribosome affinity purification technology and microarrays to
identify astrocytic genes whose expression is affected by the sleep/
wake cycle and found Mertk among the “wake” genes, upregu-
lated in both spontaneous wake and acute sleep deprivation
relative to sleep (Bellesi et al., 2015). Here the same data sets
(NCBI GEO accession number GSE69079) were interrogated by
comparing S either with forced wake or with spontaneous wake,
to determine whether among the previously identified astrocytic
transcripts involved in phagocytosis (Cahoy et al., 2008) some
were specifically affected by acute sleep loss but not by spontane-
ous wake. We found little evidence for additional activation of
phagocytic genes in forced wake relative to spontaneous wake:
Mertk showed a similar increase in both comparisons (S vs W,
p0.005; S vs SD, p0.003; Fig. 2A), and Gas6, the MERTK
ligand, also showed a similar trend toward an increase (both p
0.06; Fig. 2A). The only difference was crk, whose protein inter-
acts with DOCK1, the downstream pathway of MERTK, which
trended to increase (p0.06) only after forced wake, and Itgb2,
which instead increased ( p0.014) only after spontaneous wake
(Fig. 2A). Overall, these results suggest that a few hours of wake
are sufficient to activate the MERTK pathway, even without sleep
loss. To verify whether Mertk was upregulated also at the protein
level, we first double stained coronal sections of frontal cortex
with antibodies against MERTK and GFAP, a well recognized
marker for astrocytes. We confirmed that several astrocytic pro-
cesses were MERTK positive (Fig. 2B), as described previously
(Chung et al., 2013). Then, to assess the MERTK expression level
in nearby synapses, we prepared cortical synaptoneurosomes
from S, SD, and CSR mice, and after checking that synaptoneu-
rosomes still contained perisynaptic glia using the astrocytic
marker GFAP (Fig. 2C, top), we measured MERTK protein ex-
pression (Fig. 2C, bottom). Quantitative immunoblot analysis
showed that both SD (Dunn’s multiple comparison test, p
0.05) and CSR (Dunn’s multiple comparison test, p0.05) were
associated with higher MERTK levels relative to S, and the in-
crease was similar in the two conditions (Fig. 2D), again suggest-
ing that the activation of MERTK is linked to being awake but
does not reflect the severity and/or duration of sleep loss.
Through the action of Gas6, the MERTK receptor can recog-
nize “eat-me” signals on the membrane of the cell that needs to be
phagocyted. These signals include the exposure of phosphatidyl-
serine on the outer leaflet of the plasma membrane (Ravichan-
dran, 2010), which can occur because of oxidative stress (Kagan
et al., 2002;Brown and Neher, 2014). To assess whether sleep loss
was associated with high levels of oxidative stress at the synaptic
level, we measured the extent of lipid peroxidation by quantifying
free MDA, a lipid peroxidation end product, in cortical synap-
toneurosomes of S, SD, and CSR mice. Colorimetric quantifica-
tion of MDA levels showed a similar trend toward an increase in
SD and CSR mice relative to S mice (KW test, p0.065), whereas
Figure 2. Sleep loss is associated with MERTK upregulation. A, Heat diagram showing the expression levels of astrocytic genes previously identified (Cahoy et al., 2008) as indicative of
phagocytosis in astrocytic-enriched samples of S, W, and SD adult heterozygous Aldh1L1-eGFP-L10a mice (Bellesi et al., 2015).
p0.05 in S versus W; *p0.1 and **p0.01 in S versus SD.
B, Example of an astrocyte stained with GFAP (red) and coexpressing MERTK (green) along its processes (arrowheads). Scale bar, 30
m. C, Top, GFAP expression in cortical homogenates (HN) and
synaptoneurosomes (SYN). Bottom, Representative bands from S, SD, and CSR pools (n4 per pool) showing MERTK expression in cortical synaptoneurosomes. D, Western blot quantification of
MERTK expression in SD ( p0.05) and CSR (p0.05) relative to S. E, Lipid peroxidation analysis showing MDA concentration for S, SD, and CSR mice (KW test, p0.065).
5268 J. Neurosci., May 24, 2017 37(21):5263–5273 Bellesi et al. Glial Phagocytosis in Sleep and Wake
no difference was observed between SD and CSR (MW test, p
0.4; Fig. 2E).
Chronic sleep restriction is associated with
microglia activation
MERTK protein is also expressed in microglia (Chung et al.,
2013), and microglia contributes to synaptic elimination during
normal development (Paolicelli et al., 2011;Schafer et al., 2012;
Bialas and Stevens, 2013) and in response to monocular depriva-
tion (Sipe et al., 2016). Thus, we sought to assess whether sleep
loss leads to microglia activation in mouse cerebral cortex. We
stained S, SD, and CSR brain sections with IBA-1, a recognized
marker for microglia and quantified microglia density in the
frontal cortex (Fig. 3A). Despite a small increase in CSR relative
to SD and S mice, we found no significant changes in cell number
(KW test, p0.09; Fig. 3B). Then, we analyzed the morphology
of microglial cells, since it correlates closely with their state of
activation (Kreutzberg, 1996;Nimmerjahn et al., 2005). To quan-
tify the complexity of microglia branching, we used two different
validated approaches. The first method (Morrison and Filosa,
2013) calculates the number of process end points per cell and the
length of microglia processes per cell by skeletonizing despeckled
IBA-1-stained fields (Fig. 3C). The number of end points per cell
did not significantly change across conditions (KW test, p0.15),
although a trend toward a decrease was present in CSR relative to
S animals (MW test, p0.06; Fig. 3D). Pairwise comparisons
between groups (S vs SD, SD vs CSR) were not significant. Quan-
titative analysis showed instead a significant reduction of process
length per cell in CSR relative to S mice (MW test, p0.004; Fig.
3E). In the second approach, using a custom-made Matlab algo-
rithm based on the study by Kozlowski and Weimer (2012),we
measured the area and perimeter of automatically, individually
identified IBA-1 microglial cells. For each experimental group,
cells were clustered in quartiles (from small area/perimeter to
large area/perimeter), and the relative number of cells within
each quartile was estimated (Fig. 3F). Repeated-measures two-
way ANOVA with experimental group as the between factor and
quartiles as the within factor showed a main effect of experimen-
Figure 3. Chronic sleep loss is associated with microglia activation. A, Raw images from S (n6), SD (n5), and CSR (n6) mice (frontal cortex) showing IBA-1 staining. Scale bar, 30
B, Number of IBA-1-positive cells per cubed millimeter in S, SD, and CSR mice. C, Example of one IBA-1-positive microglial cell as it appears from the raw image and after processing (despeckling and
skeletonizing).D,E, Number of end pointsper cell (D) andsum of allprocess lengths permicroglial cell (E)in S, SD,and CSR mice.*p0.05. F, Left,Examples from Sand CSR fieldsshowing processed
and color-coded IBA-1 microglial cells (yellow, more ramified; blue, less ramified). Right, Examples of poorly ramified (above) and very ramified (below) IBA-1 microglial cells. G, Distribution in
quartiles of the number of IBA-1 microglial cells ranked by area size, an indirect measure of the complexity of process branching. *p0.05.
Bellesi et al. Glial Phagocytosis in Sleep and Wake J. Neurosci., May 24, 2017 37(21):5263–5273 • 5269
tal group (area: F
4.16, p0.038; perimeter: F
p0.05) and a significant interaction (area: F
6.82, p
0.001; perimeter: F
6.35, p0.001). Post hoc analysis found
that in the first quartile, the number of cells was higher in CSR mice
relative to S (area: Bonferroni’s test, t4.78; p0.001; perimeter:
Bonferroni’s test, t4.95; p0.001) and SD (area: Bonferroni’s
test, t4.58; p0.001; perimeter: Bonferroni’s test, t4.68;
p0.001) mice, whereas the opposite was true in the fourth
quartile (CSR vs S, area: Bonferroni’s test, t3.44, p0.01;
perimeter: Bonferroni’s test, t3.44, p0.01; CSR vs SD, area:
Bonferroni’s test, t4.06, p0.001; perimeter: Bonferroni’s
test, t3.44, p0.01). These results suggest that there was a
higher number of less ramified cells and a lower number of well
ramified cells in CSR mice relative to SD and S mice (Fig. 3G,
values for perimeter are not shown).
Furthermore, we investigated whether sleep loss promoted
microglial phagocytosis by quantifying the number and volume
of presynaptic terminals, identified as VGLUT-1-positive puncta
with confocal microscopy, which were engulfed within IBA-1-
stained cells. Only VGLUT-1-positive puncta larger than 100 pix-
els (roughly corresponding to 0.03
) and showing an overlap
of 100% in xyz with microglial cells were considered as phago-
cyted (Fig. 4A–C). Quantitative analysis showed that the number
and volume of phagocyted VGLUT-1 puncta changed signifi-
cantly across conditions (KW test; number, p0.03; volume,
p0.03). Specifically, phagocyted VGLUT-1 puncta were more
numerous in CSR mice than S mice (density, 27.98 13.56%;
MW test, p0.009) and larger in CSR mice than S mice
(32.13 22.38%; MW test, p0.026) and SD mice (38.52
23.46%; MW test, p0.03; Fig. 4D,E). The percentage of
VGLUT-1 puncta engulfed within microglia relative to the total
number of VGLUT-1 puncta was higher in CSR mice (0.39
0.14%) than S (0.22 0.07%; MW test, p0.04) and SD (0.19
0.06%, MW test, p0.017; data not shown) mice.
To further characterize microglial-mediated phagocytosis, we
measured C3 expression levels in cortical homogenates of S, SD,
and CSR mice. C3, a central component of the complement cas-
cade, is deposited on cell debris and can directly activate C3 re-
ceptors on microglia, thus triggering phagocytosis. Western blot
analysis showed that C3 expression was higher in CSR mice than
S mice (Dunn’s multiple comparison test, p0.04). Despite
some variability, SD mice also showed higher C3 levels than S
animals (Dunn’s multiple comparison test, p0.04), suggesting
that even shorter periods of sleep loss can trigger C3 activation
(Fig. 4F). Overall, these results indicate that CSR is associated
with microglia activation and increased phagocytosis.
Finally, we ascertained whether microglia activation was asso-
ciated with increased levels of inflammatory mediators in the
CSF. CSF was extracted in additional groups of S, SD, and CSR
mice. Multiplex immune assay analysis showed that 14 of the 23
molecules analyzed (i.e., IL1a, IL2, IL3, IL4, IL5, IL6, IL10, IL12,
, MIP1a, and RANTES) were undetectable in
almost all CSF samples. Nine of the remaining molecules (i.e.,
IL1b, IL9, IL12, IL13, Eotaxin, KC, MCP1, MIP1b, and TNF
were detected in 34 –100% of samples. Levels of IL1b, IL9, IL12,
IL13, Eotaxin, KC, MCP1, and MIP1b did not change signifi-
cantly across groups, whereas levels of TNF
were higher in S
mice relative to SD (MW test, p0.018) and CSR (MW test, p
0.047; Fig. 4G). Overall, these results indicate that CSR is associ-
ated with microglia activation and increased phagocytosis with-
out a notable increase of inflammatory mediators in the CSF.
We show that acute sleep deprivation and chronic sleep loss in-
crease the number of phagocytic events mediated by astrocytes in
the mouse cortical neuropil, whereas only chronic sleep loss can
trigger microglial phagocytosis. In astrocytes, phagocytosis is as-
sociated with increased MERTK expression and lipid peroxida-
tion, whereas microglial phagocytosis is associated with increased
levels of the complement component C3 without clear signs of
inflammations in CSF.
Only a few synapses are affected by AP: peripheral astrocytic
processes target 80% of all excitatory synapses, the larger ones,
and 10% of them on average undergo AP. Our adolescent mice
had already experienced the critical period of most intense syn-
aptic pruning, but more subtle synaptic refinement was likely still
occurring (Hoel et al., 2016). Early developmental phagocytosis
mainly targets presynaptic elements of transient, likely weaker,
retinogeniculate synapses (Schafer et al., 2012;Chung et al.,
2013). By contrast, wake-enhanced phagocytosis preferentially tar-
gets larger, and thus stronger, synapses and often involves axonal
elements outside the presynaptic terminal. Thus, glial phagocy-
tosis may serve different functions: elimination of exuberant syn-
apses during early development and degradation of components
of strong, likely well established synapses in response to extended
wake during adolescence. Astrocytes could promote the housekeep-
ing of worn synaptic components, especially axonal elements, by
degrading portions of their membranes, perhaps damaged by ex-
cessive lipid peroxidation. Of note, we found that the presence of
mitochondria within the presynaptic boutons did not increase
the likelihood of a synapse to be phagocyted. At first, this result
seems to exclude a direct link between oxidative energy metabo-
lism and AP. However, mitochondria are present in only 40%
of presynaptic terminals (Chavan et al., 2015;de Vivo et al., 2017)
and are rarely seen in spine heads (Sorra and Harris, 2000), which are
characterized by intense metabolic activity (Harris et al., 2012), sug-
gesting that the presence of mitochondria may be a poor marker of
the overall metabolic activity of a synapse.
Our results suggest that extended wake enhances AP through
a mechanism that involves the MERTK receptor. In fact, a few
hours of spontaneous wake are sufficient to upregulate Merkt
expression relative to sleep (Bellesi et al., 2015), but not to in-
crease the incidence of AP (this study). Thus, the activation of the
MERTK pathway may start during spontaneous wake, but its
long-term, structural consequences become apparent only after
sustained sleep loss. MERTK recognizes “eat me signals” pre-
sented in target debris (Ravichandran, 2010). One of them is
phosphatidylserine, a phospholipid normally confined to the in-
ner leaflet of the plasma membrane, which triggers phagocytosis
when exposed on the cell surface. Increased calcium concentra-
tions, ATP depletion, and oxidative stress are all factors linked to
cell activity and metabolism that can induce membrane translo-
cation of phosphatidylserine (Brown and Neher, 2014). Since
synaptic activity accounts for most of the brain’s energy budget
(Harris et al., 2012), greater metabolic activity and/or increased
production of waste induced by extended wake (Cirelli et al.,
2006) could favor the externalization of phosphatidylserine on
the plasma membrane of heavily used synapses. Another possible
mechanism involves C1q, which localizes at the sites of synaptic
elimination in the developing reticulogeniculate system (Stevens
et al., 2007). The expression of all three subunits of the C1q
complex is upregulated when retinal ganglion cells are exposed to
astrocytes, and through C3 activation, C1q can initiate synapse
elimination by the classical complement cascade (Stevens et al.,
5270 J. Neurosci., May 24, 2017 37(21):5263–5273 Bellesi et al. Glial Phagocytosis in Sleep and Wake
2007). Of note, the C1q subunit
mRNA is upregulated in the
cortex of adult rats in wake relative to sleep (Cirelli et al., 2004),
and in the current study, C3 levels increased after acute and
chronic sleep loss relative to sleep. Using the same dataset of this
study, we recently also found that synaptic density in frontal
cortex does not change between S and SD, and most spines de-
crease in size during sleep in a manner proportional to their size
(de Vivo et al., 2017). Crucially, this downscaling is diffuse but
selective, sparing the large synapses (de Vivo et al., 2017) in which
we show here that AP is more common. Thus, stronger and more
“rigid” synapses, whose strength does not seem to change be-
tween sleep and wake, may use AP to recycle structural compo-
nents and guarantee a proper synaptic function, perhaps not only
in response to damage, but to prevent it.
In addition to AP, we also found endosomes enclosed within
the PAPs. In eukaryotic cells, endosomes are involved in mem-
brane recycling, receptor trafficking, exocytosis, and cellular
waste disposal (Maxfield and McGraw, 2004). In PAPs, we can-
not exclude that some of the endosomes, those containing undi-
gested material resembling presynaptic vesicles, represent further
Figure4. Chronic sleep loss is associatedwith microglial phagocytosis. A,Raw image showingan IBA-1-positive microglia(green) and VGLUT-1puncta staining (magenta)in a representativeCSR
mouse. Scale bar, 5
m. B, Enlarged frame of the cell shown in A, visualized also in the xz and yz projections and in gray separated channels, showing a VGLUT-1-positive element engulfed within
the microglial soma (arrowheads). C, 3D reconstruction of the same cell showing the engulfed VGLUT-1 element (arrowhead). D,E, Number (D) and volume (E) of phagocyted VGLUT-1 elements per
microglial cell for S (n6), SD (n5), and CSR (n6). *p0.05. F, Western blot analysis of the complement component C3 for SD and CSR pools relative to S pools. Representative bands are
depicted above from cortical homogenates of S, SD, and CSR pools (n4 per pool). G, Protein levels of cytokines and chemokines in CSF from S (n11), SD (n10), and CSR (n8) mice. Protein
levels were measured in individual CSF specimens using multiplex magnetic bead technology for the simultaneous measurement of the 23 cytokines/chemokines. Shown is the expression of the
detected molecules and the relative pvalues obtained from the KW test.
Bellesi et al. Glial Phagocytosis in Sleep and Wake J. Neurosci., May 24, 2017 37(21):5263–5273 • 5271
steps of the phagocytosis process. On the other hand, the reduc-
tion of empty endosomes after chronic sleep loss may indicate
that they are being used during the engulfment process and/or to
recycle heavily used portions of cytoplasmic membrane. Inde-
pendent of its functional significance, which is still unclear, this
finding is in line with the results of a recent study that character-
ized ultrastructural changes in the cell body of cortical pyramidal
neurons and found that empty endosomes were more frequently
observed in S relative to CSR mice (de Vivo et al., 2016).
Microglial cells, the resident innate immune cells in the brain,
are alert sentinels of the nervous system clearing debris, looking
for signs of infiltration by infectious agents, and mediating the
inflammatory and repair response to several brain injuries (Nim-
merjahn et al., 2005;Hanisch and Kettenmann, 2007;Tay et al.,
2017). Microglia regularly extend their protrusions to briefly touch
and sense the functional state of synapses in an activity-dependent
manner (Wake et al., 2009;Tremblay et al., 2010), and their role
in developmental synaptic pruning has recently been recognized
(Paolicelli et al., 2011;Schafer et al., 2012;Bialas and Stevens,
2013;Sipe et al., 2016). The two molecular mechanisms discussed
above as potential mediators of the wake-related activation of AP
could also apply to microglia, which express both MERTK and C3
receptors (Stevens et al., 2007;Chung et al., 2013) and could be
activated by some form of damage at synaptic membranes trig-
gered by sustained neuronal activity. However, direct morpholog-
ical evidence of microglial activation with transition to an active
ameboid state, and signs of microglial phagocytosis, were only found
after chronic sleep restriction, suggesting that severe and sustained
sleep loss is required to fully engage microglia. Microglial and
astrocytic activation were reported in the rat hippocampus after
5 d of total sleep deprivation (Hsu et al., 2003). Moreover, mice
treated with the antibiotic minocycline, an inhibitor of microglial
activation, showed a reduced rebound in slow-wave activity after
3 h of sleep deprivation, prompting the authors to suggest that
microglial activation may contribute to the buildup of sleep need
during extended wake (Wisor et al., 2011). In the same study,
however, short sleep deprivation did not increase the expression
of IL1
, IL6, and TNF
in brain homogenates and decreased the
expression of CD11b, which is enriched in microglia (Wisor et al.,
2011). We found that 6 8 h of sleep deprivation, which consis-
tently trigger a sleep rebound with increased slow-wave activity
(Bellesi et al., 2015), did not lead to microglial activation. Thus, in
our experimental conditions, microglia are unlikely to play a role
in sleep homeostasis.
Microglial activation after chronic sleep loss occurred without
signs of neuroinflammation. In both animals and humans, sleep
loss has been associated with a pro-inflammatory state (Mulling-
ton et al., 2010;Hurtado-Alvarado et al., 2013), and perhaps our
CSF assay was not sensitive enough to detect mild inflammation.
Alternatively, CSF inflammation may occur only in fully blown
pathological states but not in response to sleep loss per se. Since
microglia, like astrocytes, participate in the removal of synaptic
debris, their activation during prolonged wake may represent the
physiological response of these cells to worn synapses. An alter-
native explanation, however, is suggested by the finding that sleep
promotes the clearance of amyloid-
from the interstitial space
(Xie et al., 2013) whereas sleep deprivation promotes the deposi-
tion of amyloid plaques (Lim et al., 2014), which in turn can lead
to microglia activation (Xiang et al., 2006;Halle et al., 2008;Jung
et al., 2015). Persistent microglial activation, even at a low level
(microglia priming), can lead to exaggerated and detrimental
responses to a secondary insult, further promoting pathological
states (Perry and Holmes, 2014). Thus, we speculate that by
priming microglia, chronic sleep loss may increase the brain’s
susceptibility to other forms of damage, including neurodegen-
eration (Perry and Holmes, 2014;Calcia et al., 2016), although
this idea needs to be tested directly.
Our molecular screening identified Mertk and crk transcripts,
both belonging to the MERTK pathway, as the only astrocytic
genes linked to phagocytosis and overexpressed after sleep depri-
vation relative to sleep (Bellesi et al., 2015). Yet, the current study
is correlational, and we do not know whether MERTK and C3
receptors are necessary or sufficient for sleep loss-mediated
phagocytosis. Future experiments may be able to causally link the
astrocytic and microglial molecular changes to sleep loss, for in-
stance by using Mertk/(Chung et al., 2013) and CR3/
(Schafer et al., 2012) mice.
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... CSR can lead to a range of brain deficits, including impaired attention and learning, and is associated with increased risk of neuropsychiatric disorders, but also cardiovascular diseases and metabolic alterations [4][5][6][7]. Growing evidence has demonstrated that CSR is linked to a low-grade inflammation, as reflected by increased inflammatory plasma cytokines and by the presence of other markers of inflammation in the brain, such as activation of microglia cells [8][9][10]. In addition, insufficient sleep can lead to the accumulation of intracellular reactive oxygen species (ROS) and/or reactive nitrogen species (RNS), resulting in an unbalance between the oxidant and antioxidant systems of the body [10,11]. ...
... However, other works demonstrated that IL-6 has a crucial anti-inflammatory role in local and systemic inflammatory responses by modulating levels of proinflammatory cytokines [60,61]. Our previous study showed that CSR could activate microglia without affecting the levels of cytokines in the cerebral spinal fluid [8]. Sustained microglia activation could potentially increase the brain's vulnerability to various types of damage [8]. ...
... Our previous study showed that CSR could activate microglia without affecting the levels of cytokines in the cerebral spinal fluid [8]. Sustained microglia activation could potentially increase the brain's vulnerability to various types of damage [8]. In this study, we confirmed the activation of microglia induced by CSR, but we also found an increase of IBA-1 expression in the brain of CSR mice. ...
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Background: Insufficient sleep is a serious public health problem in modern society. It leads to increased risk of chronic diseases, and it has been frequently associated with cellular oxida-tive damage and widespread low-grade inflammation. Probiotics have been attracting increasing interest recently for their antioxidant and anti-inflammatory properties. Here, we tested the ability of probiotics to contrast oxidative stress and inflammation induced by sleep loss. Methods: We administered a multi-strain probiotic formulation (SLAB51) or water to normal sleeping mice and to mice exposed to 7 days of chronic sleep restriction (CSR). We quantified protein, lipid, and DNA oxidation as well as levels of gut-brain axis hormones and pro and anti-inflammatory cytokines in the brain and plasma. Furthermore, we carried out an evaluation of microglia morphology and density in the mouse cerebral cortex. Results: We found that CSR induced oxidative stress and inflammation and altered gut-brain axis hormones. SLAB51 oral administration boosted the antioxidant capacity of the brain, thus limiting the oxidative damage provoked by loss of sleep. Moreover, it positively regulated gut-brain axis hormones and reduced peripheral and brain inflammation induced by CSR. Conclusions: Probiotic supplementation can be a possible strategy to counteract oxidative stress and inflammation promoted by sleep loss.
... Moreover, as more connections have been established between sleep disruption and desynchronisation in the internal biological clocks to diseases and mortality, it becomes increasingly critical to review new insights on the potential neurophysiology mechanisms and neural-glia connections. Sleep loss and chronic insomnia have recently been associated with the activation of glial cells, oxidative stress, prolonged neuroinflammatory responses, altered neuronal metabolism and synaptic plasticity, and acceleration of age-related processes [17,116,215,248], which will be discussed throughout this review. Understanding these mechanisms may open new avenues for future therapeutic approaches. ...
... ROS and NOS that are increased with oxidative stress) [59]. For example, under chronic sleep deprivation, rodents show microglia morphology changes characterised by an enlargement of their cell body, causing the impairment of their active surveillant homeostatic role after chronic sleep deprivation [17,98]. Microglia regulation of sleep after inflammatory insult has been recently described [190]. ...
... However, other genes or proteins more targeted towards microglia involvement in insomnia remains to be elucidated. [17,232]. ...
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According to the World Health Organization, about one-third of the population experiences insomnia symptoms, and about 10-15% suffer from chronic insomnia, the most common sleep disorder. Sleeping difficulties associated with insomnia are often linked to chronic sleep deprivation, which has a negative health impact partly due to disruption in the internal synchronisation of biological clocks. These are regulated by clock genes and modulate most biological processes. Most studies addressing circadian rhythm regulation have focused on the role of neurons, yet glial cells also impact circadian rhythms and sleep regulation. Chronic insomnia and sleep loss have been associated with glial cell activation, exacerbated neuroinflammation, oxidative stress, altered neuronal metabolism and synaptic plasticity, accelerated age-related processes and decreased lifespan. It is, therefore, essential to highlight the importance of glia-neuron interplay on sleep/circadian regulation and overall healthy brain function. Hence, in this review, we aim to address the main neurobiological mechanisms involved in neuron-glia crosstalk, with an emphasis on microglia and astrocytes, in both healthy sleep, chronic sleep deprivation and chronic insomnia.
... Besides, it was recently shown that acute and chronic sleep loss promotes the phagocytosis of astrocytes of heavily used and strong synaptic elements in response to the increased neuronal activity, which occurs during extended wakefulness. Interestingly, chronic sleep loss induces microglial activation and enhanced phagocytosis without signs of neuroinflammation [26]. Therefore, reduced work of the glymphatic system during extended sleep disruption may lead to a state of sustained microglia activation. ...
... However, there has been no mathematical description of how sleep and inflammation are linked to age-related diseases [99], how sleep parameters are linked to inflammation [25], how the sleep-immune crosstalk functions [100], and how waste clearance is linked to circadian clocks and sleep [101]. Describing such a complex interaction requires a more detailed study of sleep loss effect on promoted astrocytic phagocytosis and microglial activation in the cerebral cortex [26] with particular focus on the role of the glymphatic pathway in this scenario and in neurological disorders [102]. ...
Brain aging is a complex process involving many functions of our body and described by the interplay of a sleep pattern and changes in the metabolic waste concentration regulated by the microglial function and the glymphatic system. We review the existing modelling approaches to this topic and derive a novel mathematical model to describe the crosstalk between these components within the conceptual framework of inflammaging. Analysis of the model gives insight into the dynamics of garbage concentration and linked microglial senescence process resulting from a normal or disrupted sleep pattern, hence, explaining an underlying mechanism behind healthy or unhealthy brain aging. The model incorporates accumulation and elimination of garbage, induction of glial activation by garbage, and glial senescence by over-activation, as well as the production of pro-inflammatory molecules by their senescence-associated secretory phenotype (SASP). Assuming that insufficient sleep leads to the increase of garbage concentration and promotes senescence, the model predicts that if the accumulation of senescent glia overcomes an inflammaging threshold, further progression of senescence becomes unstoppable even if a normal sleep pattern is restored. Inverting this process by "rejuvenating the brain" is only possible via a reset of concentration of senescent glia below this threshold. Our model approach enables analysis of space-time dynamics of senescence, and in this way, we show that heterogeneous patterns of inflammation will accelerate the propagation of senescence profile through a network, confirming a negative effect of heterogeneity.
... In many animal models, acute and chronic sleep loss generally affects microglial morphology, gene expression, activation [78]. After both chronic sleep loss and/or restriction in mice, microglial activation as well as microglial and astrocytic phagocytosis of synaptic components were observed, which may be a response to higher synaptic activity associated with prolonged wakefulness [102]. The authors suggested that sleep loss promotes "housekeeping" of heavily used synapses to downscale them, but these processes might also result in enhanced susceptibility to brain damage. ...
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Sleep disturbances are widely prevalent following a traumatic brain injury (TBI) and have the potential to contribute to numerous post-traumatic physiological, psychological, and cognitive difficulties developing chronically, including chronic pain. An important pathophysiological mechanism involved in the recovery of TBI is neuroinflammation, which leads to many downstream consequences. While neuroinflammation is a process that can be both beneficial and detrimental to individuals’ recovery after sustaining a TBI, recent evidence suggests that neuroinflammation may worsen outcomes in traumatically injured patients, as well as exacerbate the deleterious consequences of sleep disturbances. Additionally, a bidirectional relationship between neuroinflammation and sleep has been described, where neuroinflammation plays a role in sleep regulation and, in turn, poor sleep promotes neuroinflammation. Given the complexity of this interplay, this review aims to clarify the role of neuroinflammation in the relationship between sleep and TBI, with an emphasis on long-term outcomes such as pain, mood disorders, cognitive dysfunctions, and elevated risk of Alzheimer’s disease and dementia. In addition, some management strategies and novel treatment targeting sleep and neuroinflammation will be discussed in order to establish an effective approach to mitigate long-term outcomes after TBI.
... It is not yet clear whether sleep disorders might precede the disease; nonetheless, prolonged sleep deprivation has been found in experimental models to be mechanistically related to proinflammatory signaling axes within the CNS, such as microglial phagocytic activation, and to impact synaptic maintenance and myelination [12,13]. ...
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Multiple sclerosis (MS) represents the most common acquired demyelinating disorder of the central nervous system (CNS). Its pathogenesis, in parallel with the well-established role of mechanisms pertaining to autoimmunity, involves several key functions of immune, glial and nerve cells. The disease’s natural history is complex, heterogeneous and may evolve over a relapsing-remitting (RRMS) or progressive (PPMS/SPMS) course. Acute inflammation, driven by infiltration of peripheral cells in the CNS, is thought to be the most relevant process during the earliest phases and in RRMS, while disruption in glial and neural cells of pathways pertaining to energy metabolism, survival cascades, synaptic and ionic homeostasis are thought to be mostly relevant in long-standing disease, such as in progressive forms. In this complex scenario, many mechanisms originally thought to be distinctive of neurodegenerative disorders are being increasingly recognized as crucial from the beginning of the disease. The present review aims at highlighting mechanisms in common between MS, autoimmune diseases and biology of neurodegenerative disorders. In fact, there is an unmet need to explore new targets that might be involved as master regulators of autoimmunity, inflammation and survival of nerve cells.
Apart from other mechanisms of action, including consequences of coagulation disturbances, hypoxia, hypoperfusion, secondary injuries, sedation, etc., it is increasingly recognized that a dysregulated immune response plays an important role in the development of cerebral dysfunction in critically ill patients. The brain was long regarded as an immune-privileged organ, protected by the blood-brain barrier, but it has now become clear that the peripheral immune system may interact with the immune cells of the brain. This “neuroinflammation” may lead to synapse pathology and loss of homeostatic microglial control resulting in cerebral dysfunction that may emerge as sickness behavior, mild fluctuating cognitive dysfunction, delirium, or even coma. Moreover, it relates to persistent cognitive decline or a deterioration in mental health status, and even accelerated development of dementia. New techniques, for example positron-emission tomography (PET), enable more detailed investigations into neuroinflammation. These techniques will facilitate future research as they may be used to monitor the trajectory of neuroinflammation or to detect neuroinflammatory endpoints in phase 2 studies with promising therapeutic interventions.
Objective: The present study aimed to systematically and quantitatively review evidence derived from both postmortem brain and PET studies to explore the pathological role of glia induced neuroinflammation in the pathogenesis of ASD, and discuss the implications of these findings in relation to disease pathogenesis and therapeutic strategies. Method: An online databases search was performed to collate postmortem studies and PET studies regarding glia induced neuroinflammation in ASD as compared to controls. Two authors independently conducted the literature search, study selection and data extraction. The discrepancies generated in these processes was resolved through robust discussions among all authors. Result: The literature search yielded the identification of 619 records, from which 22 postmortem studies and 3 PET studies were identified as eligible for the qualitative synthesis. Meta-analysis of postmortem studies reported increased microglial number and microglia density as well as increased GFAP protein expression and GFAP mRNA expression in ASD subjects as compared to controls. Three PET studies produced different outcomes and emphasized different details, with one reported increased and two reported decreased TSPO expression in ASD subjects as compared to controls. Conclusion: Both postmortem evidences and PET studies converged to support the involvement of glia induced neuroinflammation in the pathogenesis of ASD. The limited number of included studies along with the considerable heterogeneity of these studies prevented the development of firm conclusions and challenged the explanation of variability. Future research should prioritize the replication of current studies and the validation of current observations.
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Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents.
Although both nonrapid eye movement (NREM) sleep loss and rapid eye movement (REM) sleep loss exacerbate Alzheimer's disease (AD) progression, they exert different effects. Microglial activation can be beneficial or detrimental to AD patients under different conditions. However, few studies have investigated which sleep stage is the main regulator of microglial activation or the downstream effects of this activation. We aimed to explore the roles of different sleep phases in microglial activation and to investigate the possible effect of microglial activation on AD pathology. In this study, thirty-six 6-month-old APP/PS1 mice were equally divided into 3 groups: the stress control (SC), total sleep deprivation (TSD), and REM deprivation (RD) groups. All mice underwent a 48-hour intervention before their spatial memory was assessed using a Morris water maze (MWM). Then, microglial morphology, activation- and synapse-related protein expression, and inflammatory cytokine and amyloid β (Aβ) levels in hippocampal tissues were measured. We found that the RD and TSD groups exhibited worse spatial memory in the MWM tests. In addition, the RD and TSD groups showed greater microglial activation, higher inflammatory cytokine levels, lower synapse-related protein expression and more severe Aβ accumulation than the SC group, but there were no significant differences between the RD and TSD groups. This study demonstrates that disturbance of REM sleep may activate microglia in APP/PS1 mice. These activated microglia may promote neuroinflammation and engulf synapses but show a weakened ability to clear plaques.
Astrocytic fine processes are the most minor structures of astrocytes but host much of the Ca2+ activity. These localized Ca2+ signals spatially restricted to microdomains are crucial for information processing and synaptic transmission. However, the mechanistic link between astrocytic nanoscale processes and microdomain Ca2+ activity remains hazily understood because of the technical difficulties in accessing this structurally unresolved region. In this study, we used computational models to disentangle the intricate relations of morphology and local Ca2+ dynamics involved in astrocytic fine processes. We aimed to answer: 1) how nano-morphology affects local Ca2+ activity and synaptic transmission, 2) and how fine processes affect Ca2+ activity of large process they connect. To address these issues, we undertook the following two computational modeling: 1) we integrated the in vivo astrocyte morphological data from a recent study performed with super-resolution microscopy that discriminates sub-compartments of various shapes, referred to as nodes and shafts to a classic IP3R-mediated Ca2+ signaling framework describing the intracellular Ca2+ dynamics, 2) we proposed a node-based tripartite synapse model linking with astrocytic morphology to predict the effect of structural deficits of astrocytes on synaptic transmission. Extensive simulations provided us with several biological insights: 1) the width of nodes and shafts could strongly influence the spatiotemporal variability of Ca2+ signals properties but what indeed determined the Ca2+ activity was the width ratio between nodes and shafts, 2) the connectivity of nodes to larger processes markedly shaped the Ca2+ signal of the parent process rather than nodes morphology itself, 3) the morphological changes of astrocytic part might potentially induce the abnormality of synaptic transmission by affecting the level of glutamate at tripartite synapses. Taken together, this comprehensive model which integrated theoretical computation and in vivo morphological data highlights the role of the nanomorphology of astrocytes in signal transmission and its possible mechanisms related to pathological conditions.
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Microglia are the resident immune cells of the brain. Increasingly, they are recognized as important mediators of normal neurophysiology, particularly during early development. Here we demonstrate that microglia are critical for ocular dominance plasticity. During the visual critical period, closure of one eye elicits changes in the structure and function of connections underlying binocular responses of neurons in the visual cortex. We find that microglia respond to monocular deprivation during the critical period, altering their morphology, motility and phagocytic behaviour as well as interactions with synapses. To explore the underlying mechanism, we focused on the P2Y12 purinergic receptor, which is selectively expressed in non-activated microglia and mediates process motility during early injury responses. We find that disrupting this receptor alters the microglial response to monocular deprivation and abrogates ocular dominance plasticity. These results suggest that microglia actively contribute to experience-dependent plasticity in the adolescent brain.
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Rationale Psychosocial stressors are a well-documented risk factor for mental illness. Neuroinflammation, in particular elevated microglial activity, has been proposed to mediate this association. A number of preclinical studies have investigated the effect of stress on microglial activity. However, these have not been systematically reviewed before. Objectives This study aims to systematically review the effects of stress on microglia, as indexed by the histological microglial marker ionised calcium binding adaptor molecule 1 (Iba-1), and consider the implications of these for the role of stress in the development of mental disorders. Methods A systematic review was undertaken using pre-defined search criteria on PubMed and EMBASE. Inclusion and data extraction was agreed by two independent researchers after review of abstracts and full text. Results Eighteen studies met the inclusion criteria. These used seven different psychosocial stressors, including chronic restraint, social isolation and repeated social defeat in gerbils, mice and/or rats. The hippocampus (11/18 studies) and prefrontal cortex (13/18 studies) were the most frequently studied areas. Within the hippocampus, increased Iba-1 levels of between 20 and 200 % were reported by all 11 studies; however, one study found this to be a duration-dependent effect. Of those examining the prefrontal cortex, ∼75 % found psychosocial stress resulted in elevated Iba-1 activity. Elevations were also consistently seen in the nucleus accumbens, and under some stress conditions in the amygdala and paraventricular nucleus. Conclusions There is consistent evidence that a range of psychosocial stressors lead to elevated microglial activity in the hippocampus and good evidence that this is also the case in other brain regions. These effects were seen with early-life/prenatal stress, as well as stressors in adulthood. We consider these findings in terms of the two-hit hypothesis, which proposes that early-life stress primes microglia, leading to a potentiated response to subsequent stress. The implications for understanding the pathoaetiology of mental disorders and the development of new treatments are also considered.
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Astrocytes can mediate neurovascular coupling, modulate neuronal excitability, and promote synaptic maturation and remodeling. All these functions are likely to be modulated by the sleep/wake cycle, because brain metabolism, neuronal activity and synaptic turnover change as a function of behavioral state. Yet, little is known about the effects of sleep and wake on astrocytes. Here we show that sleep and wake strongly affect both astrocytic gene expression and ultrastructure in the mouse brain. Using translating ribosome affinity purification technology and microarrays, we find that 1.4 % of all astrocytic transcripts in the forebrain are dependent on state (three groups, sleep, wake, short sleep deprivation; six mice per group). Sleep upregulates a few select genes, like Cirp and Uba1, whereas wake upregulates many genes related to metabolism, the extracellular matrix and cytoskeleton, including Trio, Synj2 and Gem, which are involved in the elongation of peripheral astrocytic processes. Using serial block face scanning electron microscopy (three groups, sleep, short sleep deprivation, chronic sleep restriction; three mice per group, >100 spines per mouse, 3D), we find that a few hours of wake are sufficient to bring astrocytic processes closer to the synaptic cleft, while chronic sleep restriction also extends the overall astrocytic coverage of the synapse, including at the axon-spine interface, and increases the available astrocytic surface in the neuropil. Wake-related changes likely reflect an increased need for glutamate clearance, and are consistent with an overall increase in synaptic strength when sleep is prevented. The reduced astrocytic coverage during sleep, instead, may favor glutamate spillover, thus promoting neuronal synchronization during non-rapid eye movement sleep.
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In Alzheimer's disease (AD), hallmark ß-amyloid deposits are characterized by the presence of activated microglia around them. Despite an extensive characterization of the relation of amyloid plaques with microglia, little is known about the initiation of this interaction. In this study, the detailed investigation of very small plaques in brain slices in AD transgenic mice of the line APP-PS1(dE9) revealed different levels of microglia recruitment. Analysing plaques with a diameter of up to 10 μm we find that only the half are associated with clear morphologically activated microglia. Utilizing in vivo imaging of new appearing amyloid plaques in double-transgenic APP-PS1(dE9)xCX3CR1+/- mice further characterized the dynamic of morphological microglia activation. We observed no correlation of morphological microglia activation and plaque volume or plaque lifetime. Taken together, our results demonstrate a very prominent variation in size as well as in lifetime of new plaques relative to the state of microglia reaction. These observations might question the existing view that amyloid deposits by themselves are sufficient to attract and activate microglia in vivo.
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Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.
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Astrocytes, through their close associations with synapses, can monitor and alter synaptic function, thus actively controlling synaptic transmission in the adult brain. Besides their important role at adult synapses, in the last three decades a number of critical findings have highlighted the importance of astrocytes in the establishment of synaptic connectivity in the developing brain. In this article, we will review the key findings on astrocytic control of synapse formation, function, and elimination. First, we will summarize our current structural and functional understanding of astrocytes at the synapse. Then, we will discuss the cellular and molecular mechanisms through which developing and mature astrocytes instruct the formation, maturation, and refinement of synapses. Our aim is to provide an overview of astrocytes as important players in the establishment of a functional nervous system. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
Synapse remodeling during sleep General activity and information processing while an animal is awake drive synapse strengthening. This is counterbalanced by weakening of synapses during sleep (see the Perspective by Acsády). De Vivo et al. used serial scanning electron microscopy to reconstruct axon-spine interface and spine head volume in the mouse brain. They observed a substantial decrease in interface size after sleep. The largest relative changes occurred among weak synapses, whereas strong ones remained stable. Diering et al. found that synapses undergo changes in synaptic glutamate receptors during the sleep-wake cycle, driven by the immediate early gene Homer1a. In awake animals, Homer1a accumulates in neurons but is excluded from synapses by high levels of noradrenaline. At the onset of sleep, noradrenaline levels decline, allowing Homer1a to move to excitatory synapses and drive synapse weakening. Science , this issue p. 457 , p. 507 ; see also p. 511
Microglia are the only immune cells that permanently reside in the central nervous system (CNS) alongside neurons and other types of glial cells. The past decade has witnessed a revolution in our understanding of their roles during normal physiological conditions. Cutting-edge techniques revealed that these resident immune cells are critical for proper brain development, actively maintain health in the mature brain, and rapidly adapt their function to the physiological or pathophysiological needs. In this review, we highlight recent studies on microglial origin (from the embryonic yolk sac) and the factors regulating their differentiation and homeostasis upon brain invasion. Elegant experiments tracking microglia in the CNS allowed studies of their unique roles compared with other types of resident macrophages. Here we review the emerging roles of microglia in brain development, plasticity, and cognition, and discuss the implications of the depletion or dysfunction of microglia to our understanding of disease pathogenesis. Immune activation, inflammation, and various other conditions resulting in undesirable microglial activity at different stages of life could severely impair learning, memory, and other essential cognitive functions. The diversity of microglial phenotypes across the lifespan, between compartments of the CNS, and sexes, as well as their crosstalk with the body and external environment is also emphasised. Understanding what defines particular microglial phenotypes is of major importance for future development of innovative therapies controlling their effector functions, with consequences on cognition across chronic stress, ageing, neuropsychiatric, and neurological diseases. This article is protected by copyright. All rights reserved.
Recent evidence suggests that synaptic refinement, the reorganization of synapses and connections without significant change in their number or strength, is important for the development of the visual system of juvenile rodents. Other evidence in rodents and humans shows that there is a marked drop in sleep slow wave activity (SWA) during adolescence. Slow waves reflect synchronous transitions of neuronal populations between active and inactive states, and the amount of SWA is influenced by the connection strength and organization of cortical neurons. Here, we investigated if synaptic refinement could account for the observed developmental drop in SWA. To this end, we employed a large-scale neural model of primary visual cortex and sections of the thalamus, capable of producing realistic slow waves. In this model, we reorganized intralaminar connections according to experimental data on synaptic refinement: during pre-refinement, local connections between neurons were homogenous, while in post-refinement neurons connected preferentially to neurons with similar receptive fields and preferred orientations. Synaptic refinement led to a drop in SWA and to changes in slow wave morphology, consistent with experimental data. To test whether learning can induce synaptic refinement, intralaminar connections were equipped with spike-timing dependent plasticity (STDP). Oriented stimuli were presented during a learning period, followed by homeostatic synaptic renormalization. This led to activity-dependent refinement accompanied again by a decline in SWA. Together, these modeling results show that synaptic refinement can account for developmental changes in SWA. Thus, sleep SWA may be used to track non-invasively the reorganization of cortical connections during development.
Study Objective: The adolescent brain may be uniquely affected by acute sleep deprivation (SD) and chronic sleep restriction (CSR), but direct evidence is lacking. We used electron microscopy to examine how SD and CSR affect pyramidal neurons in frontal cortex of adolescent mice, focusing on mitochondria, endosomes, and lysosomes that together perform most basic cellular functions, from nutrient intake to prevention of cellular stress. Methods: Adolescent (1-month old) mice slept (S) or were sleep deprived (SD, with novel objects and running wheels) during the first 6-8h of the light period, chronically sleep restricted for >4 days (CSR, using novel objects, running wheels, social interaction, forced locomotion, caffeinated water), or allowed to recover sleep for ~32h after CSR (RS). Ultrastructural analysis of 350 pyramidal neurons was performed (S=82; SD=86; CSR=103; RS=79; 4-5 mice/group). Results: Several ultrastructural parameters differed in S vs. SD, S vs. CSR, CSR vs. RS, and S vs. RS, although the different methods used to enforce wake may have contributed to some of the differences between short and long sleep loss. Differences included larger cytoplasmic area occupied by mitochondria in CSR vs. S, and higher number of secondary lysosomes in CSR vs. S and RS. We also found that sleep loss may unmask inter-individual differences not obvious during baseline sleep. Moreover, using a combination of 11 ultrastructural parameters, we could predict in up to 80% of cases whether sleep or wake occurred at the single cell level. Conclusions: Ultrastructural analysis may be a powerful tool to identify which cellular organelles, and thus which cellular functions, are most affected by sleep and sleep loss.