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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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... 104 In another study, both short sleep deprivation and chronic sleep restriction were shown to increase the rate of astrocytic phagocytosis involving presynaptic parts of larger synapses. 112 The pattern of changes in lipid peroxidation in synaptoneurosomes and the expression of MERTK, the gene responsible for astrocytic phagocytosis, was also similar, and thus authors concluded this was a cleaning of overused synaptic components during wake time. 112 However, in contrast to SD, chronic sleep restriction was associated with microglial activation and phagocytosis without other overt signs of neuroinflammation. ...
... 112 The pattern of changes in lipid peroxidation in synaptoneurosomes and the expression of MERTK, the gene responsible for astrocytic phagocytosis, was also similar, and thus authors concluded this was a cleaning of overused synaptic components during wake time. 112 However, in contrast to SD, chronic sleep restriction was associated with microglial activation and phagocytosis without other overt signs of neuroinflammation. 112 Such low-level, long-term microglial activation (microglial priming) could be associated with pathologically enhanced response to other insults, thus being detrimental to the brain. ...
... 112 However, in contrast to SD, chronic sleep restriction was associated with microglial activation and phagocytosis without other overt signs of neuroinflammation. 112 Such low-level, long-term microglial activation (microglial priming) could be associated with pathologically enhanced response to other insults, thus being detrimental to the brain. 112 Those results show that alterations in morphology and functioning of glial cells might be relevant to the pathophysiology of insomnia, and further studies on this subject would be highly desirable. ...
Insomnia is a common disorder defined as frequent and persistent difficulty initiating, maintaining, or going back to sleep. A hallmark symptom of this condition is a sense of nonrestorative sleep. It is frequently associated with other psychiatric disorders, such as depression, as well as somatic ones, including immunomediated diseases. BDNF is a neurotrophin primarily responsible for synaptic plasticity and proper functioning of neurons. Due to its role in the central nervous system, it might be connected to insomnia of multiple levels, from predisposing traits (neuroticism, genetic/epigenetic factors, etc.) through its influence on different modes of neurotransmission (histaminergic and GABAergic in particular), maintenance of circadian rhythm, and sleep architecture, and changes occurring in the course of mood disturbances, substance abuse, or dementia. Extensive and interdisciplinary evaluation of the role of BDNF could aid in charting new areas for research and further elucidate the molecular background of sleep disorder. In this review, we summarize knowledge on the role of BDNF in insomnia with a focus on currently relevant studies and discuss their implications for future projects.
... Evidence suggests microglial-mediated Aβ clearance is compromised in AD [26]. Recent studies have documented the effects of sleep loss on Aβ clearance, microglial morphology, and phagocytosis [27][28][29]. Impairment of the sleep − wake cycle diminishes microglial-mediated clearance of Aβ [25]. However, it remains unclear whether sleep restoration could reprogram disease-associated microglial response and improve its Aβ clearance ability. ...
... Accumulating evidence suggests that microglia are regulated by sleep and play a role in AD pathology [25,27,29]. Our data demonstrated that sleep deficits were rescued by optogenetic stimulation of GABAergic neurons in APP mice. ...
... However, the regulatory effects of NREM sleep and SWA on innate immunity within the central nervous system were underinvestigated [2,51]. Recent studies reported that sleep loss affected microglial morphology, phagocytosis, and Aβ clearance [28,29]. This finding opened a possibility of influencing microglia, the primary innate immune cells of the brain, by boosting SWA and NREM sleep. ...
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Background Alzheimer’s disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. Methods We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. Results APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid β (Aβ) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. Conclusions In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aβ by microglia in an AD mouse model.
... Over the past decades, microglia have been increasingly implicated in the regulation of development, function and homeostasis of the CNS. 18,19 Especially due to the intimate inter-actions between neurons and microglia 20,21 and the modulated neuroimmune responses induced by sleep insufficiency, [22][23][24][25] microglia have attracted great attention in the area of sleep biology. For example, sleep disturbance results in increased levels of proinflammatory cytokines 22,26,27 and significant morphological and functional changes of microglia. ...
... 18,19 Especially due to the intimate inter-actions between neurons and microglia 20,21 and the modulated neuroimmune responses induced by sleep insufficiency, [22][23][24][25] microglia have attracted great attention in the area of sleep biology. For example, sleep disturbance results in increased levels of proinflammatory cytokines 22,26,27 and significant morphological and functional changes of microglia. 23,28 Furthermore, inhibition of microglial activity prevents sleep pressure build-up, as well as spatial memory defects following sleep deprivation. ...
... 57,58 Serving as the resident immune cells of the CNS, the activation of microglia is tightly correlated with brain states and behaviors. 21,22,24,25,59 According to previous studies, microglia can be activated by sleep deprivation and undergo a reduction in the complexity of their processes and an increase in their production of pro-inflammatory cytokines. 24,60 Even though studies have indicated that microglia show regional heterogeneity in their characteristics upon challenge, 60 our results have demonstrated that stimulation with ASD elicits similar microglial responses in various brain regions as indicated by de-ramification and decreased surface area. ...
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Sleep is a fundamental physiological condition strongly regulated by a variety of neuroanatomical and neurochemical systems. Recent studies have indicated that catch-up sleep falls short of effectiveness to counteract the negative consequences of sleep debt however, the underlying mechanisms remain poorly understood. In this study, by using various transgenic fluorescent mouse models as well as techniques including intravital two-photon imaging and immunofluorescence staining of brain sections, we have documented morphological and functional changes of microglia during acute sleep deprivation (ASD) and subsequent short-term recovery sleep (RS). In these cerebral resident immune cells, we observed sustained microglial de-ramification, reduction of process motility and enhancement of microglial phagocytosis across brain regions. Given the intimate connections between microglia activity and neuronal plasticity, we also investigated synaptic plasticity and demonstrated an accelerated elimination of dendritic spines during both ASD and subsequent RS. Furthermore, untargeted metabolomic analyses revealed extensive whole-brain metabolic changes during ASD, and that a substantial number of metabolites and pathways failed to recover within a short period of RS. It is tempting to speculate that the disturbed cerebral metabolic homeostasis contributes to the sustained microglial activation and accelerated elimination of dendritic spines during this process. This study reveals the adverse effects of sleep loss on neuroimmunomodulation and neuronal plasticity, and implicates potential mechanisms underlying how irregular sleep schedules lead to neurological disorders.
... If we do not get enough sleep, the effects of a number of processes increase, e.g., inflammation, accumulation of protein waste, excitotoxicity, etc., which rapidly deteriorate the brain. Indeed, chronic sleep loss is accompanied by astrocytic phagocytosis of synaptic elements leading to microglia activation [3,4]. Even one night without sleep leads to the accumulation of amyloid-beta (Aβ) in the brain tissue of healthy people [5,6]. ...
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The study of functions, mechanisms of generation, and pathways of movement of cerebral fluids has a long history, but the last decade has been especially productive. The proposed glymphatic hypothesis, which suggests a mechanism of the brain waste removal system (BWRS), caused an active discussion on both the criticism of some of the perspectives and our intensive study of new experimental facts. It was especially found that the intensity of the metabolite clearance changes significantly during the transition between sleep and wakefulness. Interestingly, at the cellular level, a number of aspects of this problem have been focused on, such as astrocytes–glial cells, which, over the past two decades, have been recognized as equal partners of neurons and perform many important functions. In particular, an important role was assigned to astrocytes within the framework of the glymphatic hypothesis. In this review, we return to the “astrocytocentric” view of the BWRS function and the explanation of its activation during sleep from the viewpoint of new findings over the last decade. Our main conclusion is that the BWRS’s action may be analyzed both at the systemic (whole-brain) and at the local (cellular) level. The local level means here that the neuro-glial-vascular unit can also be regarded as the smallest functional unit of sleep, and therefore, the smallest functional unit of the BWRS.
... Astrocytes are also affected by brain maturation and aging (Sun et al., 2013;Boisvert et al., 2018;Clarke et al., 2018;Lattke et al., 2021;E. Lee et al., 2022), sex (Bracchi-Ricard et al., 2008;Baier et al., 2022;Krawczyk et al., 2022;Meadows et al., 2022), and genetic variations (Messing et al., 2012;Arnaud et al., 2022), and they are sensitive to sensory processing (Schummers et al., 2008), locomotion (Paukert et al., 2014), arousal (Rasmussen et al., 2023;Reitman et al., 2023 (Bellesi et al., 2017), social isolation (Cheng et al., 2023), and viral infections (Soung and Klein, 2018;Jorgacevski and Potokar, 2023). It is possible that astrocytes play precise and context-dependent roles as a result of their integrated processing of diverse cues inherent to different biological and environmental contexts. ...
Astrocytes are emerging as key regulators of cognitive function and behavior. This review highlights some of the latest advances in the understanding of astrocyte roles in different behavioral domains across lifespan and in disease. We address specific molecular and circuit mechanisms by which astrocytes modulate behavior, discuss their functional diversity and versatility, and highlight emerging astrocyte-targeted treatment strategies that might alleviate behavioral and cognitive dysfunction in pathologic conditions. Converging evidence across different model systems and manipulations is revealing that astrocytes regulate behavioral processes in a precise and context-dependent manner. Improved understanding of these astrocytic functions may generate new therapeutic strategies for various conditions with cognitive and behavioral impairments.
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Most cognitive functions involving the prefrontal cortex emerge during late development. Increasing evidence links this delayed maturation to the protracted timeline of prefrontal development, which likely does not reach full maturity before the end of adolescence. However, the underlying mechanisms that drive the emergence and fine-tuning of cognitive abilities during adolescence, caused by circuit wiring, are still unknown. Here, we continuously monitored prefrontal activity throughout the postnatal development of mice and showed that an initial activity increase was interrupted by an extensive microglia-mediated breakdown of activity, followed by the rewiring of circuit elements to achieve adult-like patterns and synchrony. Interfering with these processes during adolescence, but not adulthood, led to a long-lasting microglia-induced disruption of prefrontal activity and neuronal morphology and decreased cognitive abilities. These results identified a nonlinear reorganization of prefrontal circuits during adolescence and revealed its importance for adult network function and cognitive processing.
Chronic sleep/wake disturbances are strongly associated with traumatic brain injury (TBI) in patients and are being increasingly recognized. However, the underlying mechanisms are largely understudied and there is an urgent need for animal models of lifelong sleep/wake disturbances. The objective of this study was to develop a chronic TBI rodent model and investigate the lifelong chronic effect of TBI on sleep/wake behavior. We performed repetitive midline fluid percussion injury (rmFPI) in four months old mice and monitored their sleep/wake behavior using the non-invasive PiezoSleep system. The sleep/wake states were recorded before injury (baseline) and then monthly thereafter. We found that TBI mice displayed a significant decrease in sleep duration in both the light and dark phases, beginning at three months post-TBI and continuing throughout the study. Consistent with the sleep phenotype, these TBI mice showed circadian locomotor activity phenotypes and exhibited reduced anxiety-like behavior. TBI mice also gained less weight, and had less lean mass and total body water content, compared to sham controls. Furthermore, TBI mice showed extensive brain tissue loss and increased GFAP and IBA1 levels in the hypothalamus and the vicinity of the injury, indicative of chronic neuropathology. In summary, our study identified a critical time window of TBI pathology and associated circadian and sleep/wake phenotypes. Future studies should leverage this mouse model to investigate the molecular mechanisms underlying the chronic sleep/wake phenotypes following TBI early in life.
Objectives This study explored the association between insomnia and the clinical outcome of large vessel occlusion Acute Ischemic Stroke (AIS) and attempted to explore its potential mechanisms from the perspectives of inflammation and oxidative stress. Methods AIS patients who underwent endovascular treatment for large vessel occlusion at Binzhou Central Hospital from 2018 to 2022 (n = 508) were included. Patients were divided into an insomnia group and a non-insomnia group. Insomnia was judged by self-reported Athens Insomnia Scale score. Regression analysis was used to compare the differences in the 24-hour and 7-day National Institutes of Health Stroke Scale (NIHSS) score, Early Neurological Deterioration (END), early adverse event incidence, 90-day prognosis and mortality, and serum biomarkers levels. Results The incidence of insomnia in the study population was 39.6% (n = 144, insomnia group; n = 364, non-insomnia group). Compared with the non-insomnia group, a worse prognosis outcome (63% vs. 49%, adjusted rate ratio: 1.8, 95% Confidence Interval: 1.2–3.7; p = 0.016), higher 24-h and 7-day NIHSS score (17 [9–36] vs. 13 [5–20]; p = 0.024, and 11 [4‒24) vs. 8 [2‒14]; p = 0.031, respectively), higher END (24% vs. 15%, p = 0.022), and higher incidence of adverse events were observed in the insomnia group (79% vs. 59%, p = 0.010). The 90-day mortality was higher in the insomnia group than that in the non-insomnia group (22% vs. 17%), however, such a difference was not statistically significant. Conclusion Insomnia is closely related to the clinical outcome of AIS with large vessel occlusion, and inflammation and oxidative stress mechanisms may be involved.
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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.